Engineered polypeptides having enhanced duration of action

ABSTRACT

Compounds are provided having inter alia good duration of action, high potency and/or convenient dosing regimens including once weekly administration. The compounds are engineered polypeptides which incorporate an albumin binding domain in combination with one or more biologically active polypeptides. Also provided are pharmaceutical compositions and methods of treatment for diseases and disorders including lipodystrophy, dyslipidemia, hyperlipidemia, overweight, obesity, hypothalamic amenorrhea, Alzheimer&#39;s disease, leptin deficiency, fatty liver disease or diabetes (including type I and type II). Additional diseases and disorders which can be treated by the compounds and methods described herein include nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver disease (NAFLD), metabolic syndrome X and Huntington&#39;s Disease.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of International Patent ApplicationNo. PCT/US2011/053786, filed Sep. 28, 2011, which claims priority toU.S. Application No. 61/387,402 filed Sep. 28, 2010, and U.S.Application No. 61/422,091 filed Dec. 10, 2010, the disclosures of whichare incorporated by reference herein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 25, 2013, isnamed 92494-867824_ST25.TXT and is 275,743 bytes in size.

BACKGROUND OF THE INVENTION

The present application relates to compounds having good duration ofaction, high potency and/or convenient dosing regimens including oraladministration, and method of use thereof. There are provided hereinengineered polypeptides which incorporate an albumin binding domain incombination with a biologically active peptide. Without wishing to bebound by any theory, it is believed that because the engineeredpolypeptides described herein can bind albumin, the compounds can besequestered (e.g., bound to albumin) while in the circulation leading toincreased duration of action, due for example to decreased renalclearance and/or degradation. Diseases amendable to such treatmentinclude lipodystrophy, dyslipidemia, hyperlipidemia, overweight,obesity, hypothalamic amenorrhea, Alzheimer's disease, leptindeficiency, fatty liver disease, diabetes (including type I and typeII), nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liverdisease (NAFLD), metabolic syndrome X and Huntington's Disease, orcombinations thereof.

There remains a need to develop polypeptides useful in the abovedescribed metabolic diseases, conditions and disorders. Accordingly, itis an object of the present invention to provide engineered polypeptideswith extended half-lives useful to treat the above conditions andmethods for producing and using them.

Each patent, patent application, and publication cited herein is herebyincorporated herein by reference in its entirety and for all purposes.

BRIEF SUMMARY OF THE INVENTION

There are provided engineered polypeptide compounds having bindingaffinity for albumin and an additional therapeutic utility. Thecompounds are engineered polypeptides which include an albumin bindingdomain (ABD) polypeptide capable of binding albumin and a hormone domain(HD) polypeptide, which HD polypeptides can be biologically active andcan elicit a beneficial biological response, in covalent linkage withthe ABD. Any of the ABD or HD polypeptides described herein can beoptionally covalently bonded in the engineered polypeptide through alinker L, for example L1 as described herein. Without wishing to bebound by any theory, it is believed that because the engineeredpolypeptides described herein can bind albumin, the compounds can besequestered in a subject leading to increased duration of action in thesubject.

In a first aspect, there is provided an engineered polypeptide asdescribed herein. The engineered polypeptide includes an albumin bindingdomain polypeptide (ABD) and a hormone domain (HD1). The hormone domainincludes a polypeptide which is a leptin, an analog of a leptin or anactive fragment thereof.

In another aspect, there is provided a method for treating a disease ordisorder in a subject in need of treatment. The method includesadministering an engineered polypeptide as described herein to thesubject.

In yet another aspect, there is provided a pharmaceutical compositionwhich includes an engineered polypeptide compound described herein incombination with a pharmaceutically acceptable excipient.

In yet another aspect are polynucleotides encoding the engineeredpolypeptide and their intermediates, expression vectors bearing suchpolynucleotides, host cells expressing such polynucleotides, and meansfor their expression, synthesis, post-translational modification andisolation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B depict the effects of a single administration of engineeredpolypeptides as described herein on food intake and body weight uponadministration to lean rats as described in Example 3. FIG. 1A: foodintake. FIG. 1B: change in body weight (% vehicle-corrected). Legend:Vehicle (box); Cmpd 1 at 2.6 mg/kg (triangle tip up); Cmpd 2 at 2.7mg/kg (triangle tip down); Cmpd 4 at 2.7 mg/kg (diamond); Cmpd C2 at 10mg/kg (circle).

FIGS. 2A-2B depict the effects of a single administration of engineeredpolypeptides as described herein on food intake and body weight uponadministration to lean rats as described in Example 4. FIG. 2A: foodintake. FIG. 2B: change in body weight (% vehicle-corrected). Legend:Vehicle (box); Cmpd 2 at 0.3 mg/kg (triangle tip up); Cmpd 2 at 1.0mg/kg (triangle tip down); Cmpd 2 at 3.0 mg/kg (diamond).

FIGS. 3A-3B depict the effects of a single administration of engineeredpolypeptides as described herein on food intake and body weight uponadministration to lean rats as described in Example 5. FIG. 3A: foodintake. FIG. 3B: change in body weight (% vehicle-corrected). Legend:Vehicle (box); Cmpd C2 at 1.1 mg/kg (circle); Cmpd C2 at 3.3 mg/kg(box); Cmpd C2 at 11.1 mg/kg (triangle tip up).

FIGS. 4A-4B depict the effects of a single administration of engineeredpolypeptides described herein, and of a control compound, on food intakeand body weight upon administration to lean rats as described in Example6. FIG. 4A: food intake. FIG. 4B: change in body weight (%vehicle-corrected). Legend: Vehicle (box); Cmpd C6 at 2.2 mg/kg(triangle tip down).

FIG. 5 depicts the effects of once weekly administration of SEQ ID NO:54on body weight (% baseline) upon administration to DIO rats as describedin Example 7. Legend: Vehicle (box); Cmpd 2 at 1.3 mg/kg per injection(triangle tip up).

FIGS. 6A-6B depict detection and quantification of plasma levels of SEQID NO:33 (FIG. 6A) and SEQ ID NO:54 (FIG. 6B) upon administration to DIOrats as described in Example 8.

FIG. 7 depicts the effects the effects of a single administration of theindicated engineered polypeptides described herein on change in bodyweight (% vehicle-corrected) upon administration to lean rats asdescribed in Example 9.

FIG. 8 depicts the effects of a single administration of the indicatedengineered polypeptides described herein on change in body weight (%vehicle-corrected) upon administration to lean rats as described inExample 10.

FIGS. 9A-9B depict the effects of a single administration of theindicated engineered polypeptides described herein on food intake andchange in body weight (% vehicle-corrected) upon administration to ratsas described in Example 11. FIG. 9A: food intake. FIG. 4B: change inbody weight (% vehicle-corrected).

FIG. 10 depicts the effects of a single administration of the indicatedengineered polypeptides described herein on food intake and change inbody weight (% vehicle-corrected) upon administration to lean rats asdescribed in Example 12.

FIG. 11A through FIG. 11C depict the effects of a single administrationof the indicated engineered polypeptides described herein on cumulativefood intake (FIG. 11A) and percent change in body weight (FIG. 11B andFIG. 11C) upon administration to lean rats as described in Example 13.

FIG. 12 depicts the leptin functional activity generated by Compound 2in the presence of albumin, as described in Example 15.

FIG. 13 illustrates a prolonged plasma concentration-versus-time profileCompound 2 in rats following a subcutaneous injection according toExample 16.

FIGS. 14A-14B illustrate a prolonged plasma concentration-versus-timeprofile Compound 15 in rats following a subcutaneous injection accordingto Example 16. FIG. 14A: plasma drug concentration (y-axis) as afunction of time in hours (x-axis); FIG. 14B: plasma drug concentration(y-axis) as a function of time in days (x-axis).

FIGS. 15A-15B depict the effects of a single administration of theindicated engineered polypeptides described herein on change in bodyweight (% vehicle-corrected) upon administration to lean rats (FIG. 15A)and ZDF rats (FIG. 15B) as described in Example 17.

FIG. 16 depicts the dose sparing effects of once weekly administrationof Compound 2 on body weight (% baseline) upon administration to leanrats as described in Example 18.

FIG. 17 is a graph depicting the effect on body weight of administrationof leptin (125 μg/kg/day) and amylin (1500 μg/kg/day), either alone orin combination, in two groups of rats: one group of very obese rats andanother group that was calorie restricted down to the range of moderateobesity.

FIG. 18A is a graph depicting an effect on body weight of administrationof Compound 2 (120 nmol/kg) and infused rat amylin (50 μg/kg/day),either alone or in combination over four weeks. FIG. 18B is a graphdepicting an effect on body weight of administration of Compound 2 (120nmol/kg) and PEG-rat amylin (Des-Lys1-[Lys26(mPEG40K)]-Rat Amylin (SEQID NO: 148) (125 nmol/kg), either alone or in combination over fourweeks.

FIGS. 19A-19B depict an effect on food intake (FIG. 19A) and body weight(FIG. 19B) of administration of Compound 15 (120 nmol/kg) and amylin (50μg/kg/day), either alone or in combination over four weeks.

FIG. 20 is a graph depicting an effect on body weight of administrationof Compound 15 (120 nmol/kg) and PEG-rat amylin(Des-Lys1-[Lys26(mPEG40K)]-Rat Amylin (SEQ ID NO: 148) (125 nmol/kg),either alone or in combination over four weeks.

FIG. 21A depicts an effect on body weight of administration of leptinand amylin, either alone or in combination over four weeks, inmoderately obese rats. FIG. 21B depicts the lack of an effect on bodyweight of administration of leptin and amylin, either alone or incombination over four weeks, in severely obese rats. FIG. 21C depicts aneffect on body weight of administration of Compound 2 (120 nmol/kg) andPEG-rat amylin (Des-Lys1-[Lys26(mPEG40K)]-Rat Amylin (SEQ ID NO: 148)(125 nmol/kg), either alone or in combination over four weeks, inseverely obese rats.

FIGS. 22A-22B depict an effect on body weight of administration of:(FIG. 22A) Compound 15 (120 nmol/kg) or (FIG. 22B) Compound 2 (120nmol/kg) and amylin (50 μg/kg/day), either alone or in combination overfour weeks, in severely obese rats.

FIGS. 23A-23B depict the effects of the indicated engineeredpolypeptides described herein on blood glucose upon administration toSTZ-induced T1DM mice as described in Example 30. FIG. 23A: bloodglucose (y-axis) as a function of time (x-axis); FIG. 23B: histogram ofchange in blood glucose from baseline at week 2 for vehicle, Cmpd 15,Cmpd2, and insulin.

FIGS. 24A-24B depict the effects of the indicated engineeredpolypeptides described herein on Hemoglobin A1C upon administration toSTZ-induced T1DM mice as described in Example 30. FIG. 24A: HbA1c(y-axis) as a function of time (x-axis); FIG. 24B: histogram of changein HbA1c from baseline at week 2 for vehicle, Cmpd 15, Cmpd 2, andinsulin.

FIGS. 25A-25B depict the effects of the indicated engineeredpolypeptides described herein on food intake and body weight uponadministration to STZ-induced T1DM mice as described in Example 30. FIG.25A: body weight (y-axis) as a function of time (x-axis) for vehicle,Cmpd 15, Cmpd 2, insulin, and normal; FIG. 25B: cumulative food intake(y-axis) as a function of time (x-axis) for vehicle, Cmpd 15, Cmpd 2,insulin, and normal.

FIGS. 26A-26B depict the effects of the indicated engineeredpolypeptides described herein, with and without a low dose of insulin,on blood glucose upon administration to STZ-induced T1DM mice asdescribed in Example 30. FIG. 26A: blood glucose (y-axis) as a functionof time (x-axis); FIG. 26B: histogram of blood glucose change frombaseline at week 2 for vehicle/placebo, vehicle/insulin, Cmpd15/placebo, and Cmpd15/insulin.

FIGS. 27A-27B depict the effects of the indicated engineeredpolypeptides described herein, with and without a low dose of insulin,on Hemoglobin A1C upon administration to STZ-induced T1DM mice asdescribed in Example 30. FIG. 27A: HbA1c (y-axis) as a function of time(x-axis); FIG. 27B: HbA1c percent change from baseline at week 2 forvehicle/placebo, vehicle/insulin, Cmpd 15/placebo, and Cmpd 15/insulin.

FIGS. 28A-28B depict the effects of the indicated engineeredpolypeptides described herein, with and without a low dose of insulin,on food intake (% vehicle-corrected) and change in body weight (%vehicle-corrected) upon administration to STZ-induced T1DM mice asdescribed in Example 30. FIG. 28A: cumulative food intake (y-axis) as afunction of time α-axis); FIG. 28B: percent body weight change frombaseline (y-axis) as a function of time α-axis).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

“Obesity” and “overweight” refer to mammals having a weight greater thannormally expected, and may be determined by, e.g., physical appearance,body mass index (BMI) as known in the art, waist-to-hip circumferenceratios, skinfold thickness, waist circumference, and the like. TheCenters for Disease Control and Prevention (CDC) define overweight as anadult human having a BMI of 25 to 29.9; and define obese as an adulthuman having a BMI of 30 or higher. Additional metrics for thedetermination of obesity exist. For example, the CDC states that aperson with a waist-to-hip ratio greater than 1.0 is overweight.

“Lean body mass” refers to the fat-free mass of the body, i.e., totalbody weight minus body fat weight is lean body mass. Lean body mass canbe measured by methods such as hydrostatic weighing, computerizedchambers, dual-energy X-ray absorptiometry, skin calipers, magneticresonance imaging (MRI) and bioelectric impedance analysis (BIA) asknown in the art.

“Mammal” refers to warm-blooded animals that generally have fur or hair,that give live birth to their progeny, and that feed their progeny withmilk. Mammals include humans; companion animals (e.g., dogs, cats); farmanimals (e.g., cows, horses, sheep, pigs, goats); wild animals; and thelike. In one embodiment, the mammal is a female. In one embodiment, themammal is a female human. In one embodiment, the mammal is a cat or dog.In one embodiment, the mammal is a diabetic mammal, e.g., a human havingtype 2 diabetes. In one embodiment, the mammal is an obese diabeticmammal, e.g., an obese mammal having type 2 diabetes. The term “subject”in the context of methods described herein refers to a mammal.

“Fragment” in the context of polypeptides refers herein in the customarychemical sense to a portion of a polypeptide. For example, a fragmentcan result from N-terminal deletion or C-terminal deletion of one ormore residues of a parent polypeptide, and/or a fragment can result frominternal deletion of one or more residues of a parent polypeptide.“Fragment” in the context of an antibody refers to a portion of anantibody which can be linked to a biologically active molecule tomodulate solubility, distribution within a subject, and the like. Forexample, leptin A200 described herein is a conjugate of an Fc antibodyfragment with a leptin, as known in the art. See e.g. WO 98/28427 andUS2007/002084. The term “parent” in the context of polypeptides refers,in the customary sense, to a polypeptide which serves as a referencestructure prior to modification, e.g., insertion, deletion and/orsubstitution. The term “conjugate” in the context of engineeredpolypeptides described herein refers to covalent linkage betweencomponent polypeptides, e.g., ABD, HD1 and the like. The term “fusion”in the context of engineered polypeptides described herein refers tocovalent linkage between component polypeptides, e.g., ABD, HD1 and thelike, via either or both terminal amino or carboxy functional group ofthe peptide backbone. Engineered polypeptides can be synthetically orrecombinantly made. Typically, fusions are made using recombinantbiotechnology, however, can also be made by chemical synthesis andconjugation methods known in the art.

“Analog” as used herein in the context of polypeptides refers to acompound that has insertions, deletions and/or substitutions of aminoacids relative to a parent compound. An analog may have superiorstability, solubility, efficacy, half-life, and the like. In someembodiments, an analog is a compound having at least 50%, for example50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or even higher,sequence identity to the parent compound.

“Identity,” “sequence identity” and the like in the context of comparingtwo or more nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 50% identity, preferably 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higheridentity over a specified region, when compared and aligned for maximumcorrespondence over a comparison window or designated region) asmeasured using a sequence comparison algorithms as known in the art, forexample BLAST or BLAST 2.0. This definition includes sequences that havedeletions and/or additions, as well as those that have substitutions, aswell as naturally occurring, e.g., polymorphic or allelic variants, andman-made variants. In preferred algorithms, account is made for gaps andthe like, as known in the art. For sequence comparison, typically onesequence acts as a reference sequence, to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are entered into a computer, subsequence coordinates aredesignated if necessary, and sequence algorithm program parameters aredesignated. Preferably, default program parameters can be used, oralternative parameters can be designated. The sequence comparisonalgorithm then calculates the percent sequence identities for the testsequences relative to the reference sequence, based on the programparameters. Optimal alignment of sequences for comparison can beconducted, e.g., by the local homology algorithm of Smith & Waterman,1981, Adv. Appl. Math. 2:482, by the homology alignment algorithm ofNeedleman & Wunsch, 1970, J. Mol. Biol. 48:443, by the search forsimilarity method of Pearson & Lipman, 1988, Proc. Nat'l. Acad. Sci. USA85:2444, by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manualalignment and visual inspection. See e.g., Current Protocols inMolecular Biology (Ausubel et al., eds. 1995 supplement)). Preferredexamples of algorithms that are suitable for determining percentsequence identity and sequence similarity include the BLAST and BLAST2.0 algorithms, which are described in Altschul et al., 1977, Nuci.Acids Res. 25:3389-3402 and Altschul et al., 1990, J. Mol. Biol.215:403-410. BLAST and BLAST 2.0 are used, as known in the art, todetermine percent sequence identity for the nucleic acids and proteinsof the invention. Software for performing BLAST analyses is publiclyavailable through the web site of the National Center for BiotechnologyInformation. This algorithm involves first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., Id.). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are extended in both directions along each sequencefor as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, e.g., for nucleotide sequences,the parameters M (reward score for a pair of matching residues;always >0) and N (penalty score for mismatching residues; always <0).For amino acid sequences, a scoring matrix is used to calculate thecumulative score. Extension of the word hits in each direction arehalted when: the cumulative alignment score falls off by the quantity Xfrom its maximum achieved value; the cumulative score goes to zero orbelow, due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W, T, and X determine the sensitivity and speed ofthe alignment. The BLASTN program (for nucleotide sequences) uses asdefaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=−4 anda comparison of both strands. For amino acid sequences, the BLASTPprogram uses as defaults a wordlength of 3, and expectation (E) of 10,and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, 1989, Proc.Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of10, M=5, N=−4, and a comparison of both strands.

The term “about” in the context of a numeric value refers to +/−10% ofthe numeric value, unless expressly indicated otherwise.

The terms “peptide” and “polypeptide” in the context of components ofthe engineered polypeptides described herein are synonymous.

II. Compounds

In a first aspect, engineered polypeptide compounds are provided whichinclude an albumin binding domain (ABD) polypeptide and at least onepolypeptide hormone domain (HD1). The terms “albumin binding domain,”“ABD” and the like refer to polypeptides capable of binding albumin asdescribed herein. The terms “hormone domain,” “hormone domainpolypeptide” and the like refer to a polypeptide capable of eliciting abiological response in a subject. Exemplary hormone domains include, butare not limited to, a leptin, an analog of a leptin or an activefragment thereof, but could be a leptin derivative such as a PEGylatedderivative.

It was surprisingly found that a leptin, a leptin analog, a activeleptin fragment, or a leptin derivative thereof can be fused to anvery-high-affinity albumin binding domain (ABD) derived from thealbumin-binding domains of bacterial proteins as described herein, whileretaining sufficient leptin biological activity and having an extendedduration of action, for example of at least 3 days and even 5 days in arodent, which translates to at least a one week duration or longer in ahuman subject. This was surprising in part because such ABD peptideshave not been extensively demonstrated to be a robust platform as atherapeutic protein carrier, they are relatively hydrophobic which couldinteract adversely with an attached therapeutic peptide, and were notable to act as a carrier for at least one family of peptide hormones.For instance, rat amylin compounds (e.g., SEQ ID NO:108), whenconjugated or fused to the ABDs described herein, did not display anysignificant or long-acting in vivo activity in the same rodent models inwhich various leptin engineered polypeptide constructs of the inventionwere found to be active and with long duration of action.

Biologically Active Components.

Biologically active compound components contemplated for use in thecompounds and methods described herein include leptins. The terms“biologically active compound” and the like refer in the customary senseto compounds, e.g., polypeptides and the like, which can elicit abiological response.

Leptins.

“Leptins” and “a leptin” means: leptins, leptin active fragments, leptinanalogs, and leptin derivatives; and a leptin, a leptin active fragment,a leptin analog, and a leptin derivative; respectfully. Accordingly,unless otherwise noted, reference to “leptins” is meant to leptins,leptin active fragments, leptin analogs, and leptin derivatives, asdisclosed herein. Similarly, unless otherwise noted, reference to “aleptin” is meant to encompass a leptin, a leptin active fragment, aleptin analog, and a leptin derivative, as disclosed herein. Exemplarysuch leptins which may be employed in the design, preparation, and useof the engineered polypeptides disclosed herein include those whichelicit one or more biological responses known in the art to be elicitedwhen leptins are administered to subjects (see, e.g., published U.S.Patent application Nos. US 2007/0020284 and US 2008/0207512, U.S. Pat.Nos. 6,309,853, and 7,183,254, and PCT Published Application Nos. WO96/005309, WO 98/28427, and WO 2009/064298), such as: reduction of foodintake, reduction of body weight, reduction of body weight gain,induction of satiety, reduction of caloric availability, reduction ofcaloric efficiency, reduction of metabolic plateau, increase in insulinsensitivity, reduction of hyperlipidemia, correction of dyslipidemia,reduction of hypertriglyceridemia, amelioration of obesity, ameliorationof overweight, amelioration of diabetes mellitus (including type Idiabetes, type II diabetes, and gestational diabetes), amelioration ofinsulin resistance, amelioration of lipodystrophy conditions associatedtherewith, as well as other biological responses known in the art to beelicited upon administration of a leptin (see, e.g., published U.S.Patent Application Nos. US 2007/0020284 and US 2008/0207512, U.S. Pat.Nos. 6,309,853, and U.S. Pat. No. 7,183,254, and PCT PublishedApplication Nos. WO 96/005309, WO 98/28427, and WO 2009/064298.

Exemplary leptins suitable for the design, preparation, and use of theengineered polypeptides described herein include, but are not limitedto, the compounds described in U.S. Pat. Nos. 5,594,101, 5,851,995,5,691,309, 5,580,954, 5,554,727, 5,552,523, 5,559,208, 5,756,461,6,309,853, published U.S. Patent application No. US 2007/0020284, andPCT Published Application Nos. WO 96/23517, WO 96/005309, WO 98/28427,WO 2004/039832, WO 98/55139, WO 98/12224, and WO 97/02004, each of whichis incorporated herein in its entirety and for all purposes. Methods toassay for leptin activities and biological responses in vitro and invivo, including satiety, food intake inhibition activity and weight lossactivity, are known in the art and are described herein and also in theabove references and other references recited herein.

Any leptin, leptin analog, leptin active fragment, or leptin derivativeknown in the art may be employed in order to prepare and use engineeredpolypeptides as disclosed herein throughout. Representative leptins,leptin analogs, leptin active fragments, and leptin derivativescontemplated for use in the engineered polypeptides and methodsdescribed herein also include the following:

Mature Murine Leptins:

(SEQ ID NO: 1)VPIQKVQDDTKTLIKTIVTRINDISHT-Xaa-SVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPGC, wherein Xaa at position 28 is Q or absent.

Mature Murine Leptin Form 1:

(SEQ ID NO: 143)VPIQKVQDDTKTLIKTIVTRINDISHTQSVSAKQRVTGLDFIPGLHPILSLSKMDQTLAVYQQVLTSLPSQNVLQIANDLENLRDLLHLLAFSKSCSLPQTSGLQKPESLDGVLEASLYSTEVVALSRLQGSLQDILQQLDVSPEC.

Mature Murine Leptin Form 2:

(SEQ ID NO: 144) VPIQKVQDDTKTLIKTIVTRINDISHTSVSAKQRVTGLDFIPGLHPILSLSKMDQTLAVYQQVLTSLPSQNVLQIANDLENLRDLLHLLAFSKSCSLPQTSGLQKPESLDGVLEASLYSTEVVALSRLQGSLQDILQQLDVSPEC.

Mature Murine Leptins with N-Terminal Methionine:

(SEQ ID NO: 2) MVPIQKVQDDTKTLIKTIVTRINDISHT-Xaa-SVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPGC, wherein Xaa at position 29 is Q or absent.

Mature Murine Leptin Form 1 with N-Terminal Methionine:

(SEQ ID NO: 145) MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSAKQRVTGLDFIPGLHPILSLSKMDQTLAVYQQVLTSLPSQNVLQIANDLENLRDLLHLLAFSKSCSLPQTSGLQKPESLDGVLEASLYSTEVVALSRLQGSLQDILQQLDVSPEC.

Mature Murine Leptin Form 2 with N-Terminal Methionine:

(SEQ ID NO: 146) MVPIQKVQDDTKTLIKTIVTRINDISHTSVSAKQRVTGLDFIPGLHPILSLSKMDQTLAVYQQVLTSLPSQNVLQIANDLENLRDLLHLLAFSKSCSLPQTSGLQKPESLDGVLEASLYSTEVVALSRLQGSLQDILQQLDVSPEC.

Mature Porcine Leptin:

(SEQ ID NO: 3) VPIWRVQDDTKTLIKTIVTRISDISHMQSVSSKQRVTGLDFIPGLHPVLSLSKMDQTLAIYQQILTSLPSRNVIQISNDLENLRDLLHLLASSKSCPLPQARALETLESLGGVLEASLYSTEVVALSRLQGALQDMLRQLDLSPGC.

Mature Porcine Leptin with N-Terminal Methionine:

(SEQ ID NO: 4) MVPIWRVQDDTKTLIKTIVTRISDISHMQSVSSKQRVTGLDFIPGLHPVLSLSKMDQTLAIYQQILTSLPSRNVIQISNDLENLRDLLHLLASSKSCPLPQARALETLESLGGVLEASLYSTEVVALSRLQGALQDMLRQLDLSPGC.

Mature Bovine Leptins:

(SEQ ID NO: 5) VPICKVQDDTKTLIKTIVTRINDISHT-Xaa-SVSSKQRVTGLDFIPGLHPLLSLSKMDQTLAIYQQILTSLPSRNVVQISNDLENLRDLLHLLAASKSCPLPQVRALESLESLGVVLEASLYSTEVVALSRLQGSLQDMLRQLDLSPGC, wherein Xaa at position 28 is Q or absent.

Mature Bovine Leptins with N-Terminal Methionine:

(SEQ ID NO: 6) MVPICKVQDDTKTLIKTIVTRINDISHT-Xaa-SVSSKQRVTGLDFIPGLHPLLSLSKMDQTLAIYQQILTSLPSRNVVQISNDLENLRDLLHLLAASKSCPLPQVRALESLESLGVVLEASLYSTEVVALSRLQGSLQDMLRQLDLSPGC, wherein Xaa at position 29 is Q or absent.

Unprocessed Full-Length Human Leptin (i.e., Includes 21-ResidueN-Terminal Signal Sequence):

(SEQ ID NO: 7) MHWGTLCGFLWLWPYLFYVQAVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGY STEVVALSR LQGSLQDMLWQLDLSPGC

Mature Human Leptins (with N-Terminal 21 Amino Acid Signal SequenceRemoved):

(SEQ ID NO: 8) VPIQKVQDDTKTLIKTIVTRINDISH-Xaa-Xaa-SVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC, wherein: Xaa at position 27 is T or A; andXaa at position 28 is Q or absent.

Mature Human leptins with N-terminal methionine:

(SEQ ID NO: 9) MVPIQKVQDDTKTLIKTIVTRINDISH-Xaa-Xaa-SVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC, wherein: Xaa at position 28 is T or A; andXaa at position 29 is Q or absent.

Mature Rhesus Leptin:

(SEQ ID NO: 10) VPIQKVQSDTKTLIKTIVTRINDISHTQSVSSKQRVTGLDFIPGLHPVLTLSQMDQTLAIYQQILINLPSRNVIQISNDLENLRDLLHLLAFSKSCHLPLASGLETLESLGDVLEASLYSTEVVALSRLQGSLQDMLWQLDLSPGC.

Mature Rhesus Lentin with N-Terminal Methionine:

(SEQ ID NO: 11) MVPIQKVQSDTKTLIKTIVTRINDISHTQSVSSKQRVTGLDFIPGLHPVLTLSQMDQTLAIYQQILINLPSRNVIQISNDLENLRDLLHLLAFSKSCHLPLASGLETLESLGDVLEASLYSTEVVALSRLQGSLQDMLWQLDLSPGC.

Mature Rat Leptin:

(SEQ ID NO: 12) VPIHKVQDDTKTLIKTIVTRINDISHTQSVSARQRVTGLDFIPGLHPILSLSKMDQTLAVYQQILTSLPSQNVLQIAHDLENLRDLLHLLAFSKSCSLPQTRGLQKPESLDGVLEASLYSTEVVALSRLQGSLQDILQQLDLSPEC.

Mature Rat Leptin with N-Terminal Methionine:

(SEQ ID NO: 13) MVPIHKVQDDTKTLIKTIVTRINDISHTQSVSARQRVTGLDFIPGLHPILSLSKMDQTLAVYQQILTSLPSQNVLQIAHDLENLRDLLHLLAFSKSCSLPQTRGLQKPESLDGVLEASLYSTEVVALSRLQGSLQDILQQLDLSPEC.

Mature Platypus Leptin: The Mature Platypus Leptin Sequence Follows:

(SEQ ID NO: 14)ISIEKIQADTKTLTKTIITRIIQLSTQNGVSTDQRVSGLDFIPGNQQFQNLADMDQTLAVYQQILSSLPMPDRTQISNDLENLRSLFALLATLKNCPFTRSDGLDTMEIWGGIVEESLYSTEVVTLDRLRKSLKNIEKQLDHIQG.

Unprocessed Full-Length Platypus Leptin (i.e., Includes 21-ResidueN-Terminal Signal Sequence):

A full length sequence of platypus leptin, including a 21-residueN-terminal signal sequence follows:

(SEQ ID NO: 15)MRCILLYGFLCVWQHLYYSHPISIEKIQADTKTLTKTIITRIIQLSTQNGVSTDQRVSGLDFIPGNQQFQNLADMDQTLAVYQQILSSLPMPDRTQISNDLENLRSLFALLATLKNCPFTRSDGLDTMEIWGGIVEESLYSTEVVTLDRLRKSLKNIEKQLDHIQG.

Mature Human Leptin Form 1:

(SEQ ID NO: 16)VPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC.

Mature Human Leptin Form 2:

(SEQ ID NO: 17)VPIQKVQDDTKTLIKTIVTRINDISHAQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC.

Mature Human Leptin Form 3:

(SEQ ID NO: 18)VPIQKVQDDTKTLIKTIVTRINDISHTSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC.

Mature Human Leptin Form 4:

(SEQ ID NO: 19)VPIQKVQDDTKTLIKTIVTRINDISHASVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC.

Mature Human Leptin Form 1 with N-Terminal Methionine (Also Known asMetreleptin, or A100):

(SEQ ID NO: 20)MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC.

Mature Human Leptin Form 2 with N-Terminal Methionine:

(SEQ ID NO: 21)MVPIQKVQDDTKTLIKTIVTRINDISHAQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC.

Mature Human Leptin Form 3 with N-Terminal Methionine:

(SEQ ID NO: 22)MVPIQKVQDDTKTLIKTIVTRINDISHTSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC.

Mature Human Leptin Form 4 with N-Terminal Methionine:

(SEQ ID NO: 23)MVPIQKVQDDTKTLIKTIVTRINDISHASVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC.

Seal Leptin:

(SEQ ID NO: 24)PIQRVQDDTKTLIKTIITRINDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRSVVQIANDLANLRALLRLLASAKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQDMLRQLDRNPGC.

Seal Leptin with Amino Acids 71-92 Replaced with Amino Acids 73-94(Helix 3) of Metreleptin, Respectively:

(SEQ ID NO: 25)PIQRVQDDTKTLIKTIITRINDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQDMLRQLDRNPGC.

Seal Leptin with Amino Acids 30 and 71-92 Replaced with Amino Acids 32and 73-94 (Helix 3) of Metreleptin, Respectively:

(SEQ ID NO: 26)PIQRVQDDTKTLIKTIITRINDISPPQGVSSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQDMLRQLDRNPGC.

Seal Leptin with N-Terminal Methionine:

(SEQ ID NO: 27)MPIQRVQDDTKTLIKTIITRINDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRSVVQIANDLANLRALLRLLASAKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQDMLRQLDRNPGC.

Seal Leptin with N-Terminal Methionine, and with Amino Acids 71-92Replaced with amino acids 73-94 (helix 3) of metreleptin, respectively:

(SEQ ID NO: 28)MPIQRVQDDTKTLIKTIITRINDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQDMLRQLDRNPGC.

Seal leptin with N-terminal methionine, and with amino acids 30 and71-92 Replaced with Amino Acids 32 and 73-94 (Helix 3) of Metreleptin,Respectively:

(SEQ ID NO: 29)MPIQRVQDDTKTLIKTIITRINDISPPQGVSSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQDMLRQLDRNPGC.

Leptin A200:

Leptin A200 is an Fc antibody fragment condensation product with leptin,as known in the art. See e.g., Lo et al., 2005, Protein Eng. Design &Selection, 18:1-10. The amino acid sequence of A200 is as follows:

(SEQ ID NO: 30) MDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC

Leptin A300:

Leptin A300 is metreleptin with substitutions W101Q and W139Q(N-terminal ¹Met counted as residue 1):

(SEQ ID NO: 31) MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPGC.

Leptin A400:

Leptin A400 is metreleptin with the serine residue at position 78replaced with a cysteine residue, as set forth following:

(SEQ ID NO: 32) MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQICNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC;to which a 20 kilodalton (kDa) PEG moiety has beenattached via the cysteine residue at position 78.

Leptin A500:

Research by a number of investigators including the inventors hasfocused on the effects on aggregation of residue substitution in leptin.See e.g., Ricci et al., 2006. “Mutational approach to improve physicalstability of protein therapeutics susceptible to aggregation: Role ofaltered conformation in irreversible precipitation,” Book Chapter. In:MISBEHAVING PROTEINS: PROTEIN (MIS)FOLDING, AGGREGATION, AND STABILITY,Murphy R M, Tsai A M, Eds., New York. Springer. pp. 331-350, which isincorporated herein by reference and for all purposes. Accordingly,leptin A500 with sequence following has been used in certain compoundsand methods described herein:

(SEQ ID NO: 33) MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPGC.

Leptin A100 Variants:

Variants of Leptin A100 with the following residue substitutions follow:

D41E, H98S, W101Q, D109E, G113E, M137I, W139Q and G146E:(SEQ ID NO: 664) MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCSLPQASGLETLESLGEVLEASGYSTEVVALSRLQGSLQDILQQLDLSPEC.H98S, W101Q, A102T, G113E, M137I, W139Q, and G146E: (SEQ ID NO: 665)MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCSLPQASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDILQQLDLSPEC.H98S, W101Q, G113E, M137I, W139Q, and G146E: (SEQ ID NO: 666)MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCSLPEQASGLETLDSLGEVLEASGYSTVVALSRLQGSLQDILQQLDLSPEC.W101Q, G113E, M137I, W139Q, and G146E: (SEQ ID NO: 667)MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLDSLGEVLEASGYSTEVVALSRLQGSLQDILQQLDLSPEC.H98S, W101Q, M137I, W139Q, and G146E: (SEQ ID NO: 668)MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCSLPQASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDILQQLDLSPEC.W101Q, G113E, M137I, W139Q, L143V, and G146E: (SEQ ID NO: 669)MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLDSLGEVLEASGYSTEVVALSRLQGSLQDILQQLDVSPEC.H98S, W101Q, A102T, Ml37I, W139Q, and G146E: (SEQ ID NO: 670)MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCSLPQTSGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDILQQLDLSPEC.H98S, W101Q, D109E, G113E, and G146E: (SEQ ID NO: 671)MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCSLPQASGLETLESLGEVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPEC.W101Q, M137I, W139Q, and G146E: (SEQ ID NO: 672)MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDILQQLDLSPEC.W101Q, M137I, W139Q, L143V, and G146E: (SEQ ID NO: 673)MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDILQQLDVSPEC.H98S, W101Q, A102T, M137I, W139Q, L143V, and G146E: (SEQ ID NO: 674)MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCSLPQTSGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDILQQLDVSPEC.H98S, W101Q, A102T, G113E, and G146E: (SEQ ID NO: 675)MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCSLPQTSGLETLDSLGEVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPEC.W101Q, G113E, and W139Q: (SEQ ID NO: 676)MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLDSLGEVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPGC.W101Q, G113E, W139Q, and G146E: (SEQ ID NO: 677)MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLDSLGEVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPEC.

Any of the above leptins, leptin analogs or their active fragments, aswell as leptins as described below, are suitable for use in the presentengineered polypeptides, with or without a linker to the ABD.

Albumin Binding Domain (ABD) Peptides.

Albumin binding domain (ABD) peptides for use in the invention are thosewith comparably high affinity for albumin and derive fromalbumin-binding domains of bacterial protein G of Streptococcus strainG148. As such, ABD peptides contemplated for the engineered polypeptidesdescribed herein include those having the albumin binding motifs asdescribed by Jonsson et al. (Protein Eng. Design & Selection, 2008,21:515-527) as well as the ABD peptides described therein, and thosemotifs and ABD peptides further described in PCT Published Appl. No.WO2009/016043, as well as analogs thereof, particularly those having atleast 85% amino acid identity. In one embodiment the ABD peptide cancomprise an albumin binding motif (“ABM”) that consists of the aminoacid sequence:

GVSD X₅ YK X₈ X₉ I X₁₁ X₁₂ A X₁₄ TVEGV X₂₀ AL X₂₃ X₂₄ X₂₅ I (SEQ IDNO:34)

wherein, independently of each other,

X₅ is selected from Y and F;

X₈ is selected from N, R and S;

X₉ is selected from V, I, L, M, F and Y;

X₁₁ is selected from N, S, E and D;

X₁₂ is selected from R, K and N;

X₁₄ is selected from K and R;

X₂₀ is selected from D, N, Q, E, H, S, R and K;

X₂₃ is selected from K, I and T;

X₂₄ is selected from A, S, T, G, H, L and D; and

X₂₅ is selected from H, E and D.

In certain embodiments, X₅ is Y. In certain embodiments, X₈ is N. Incertain embodiments, X₂₃ is T. In certain embodiments, X₂₃ is I. Incertain embodiments, X₂₄ is S. In certain embodiments, X₂₄ is L. Incertain embodiments, X₂₅ is E. In certain embodiments, X₂₅ is H. Incertain embodiments, independently from each other, X₅ is Y, and/or X₈is N, and/or X₂₃ is T or I, and/or X₂₄ is S or L, and/or X₂₅ is E. Incertain embodiments, the albumin binding motif (“ABM”) isGVSDYYKNLINKAKTVEGVEALTLHI (SEQ ID NO:114). In certain embodiments, thealbumin binding motif (“ABM”) is GVSDYYKNLINKAKTVEGVEALISEI (SEQ IDNO:115).

Preferably the ABD peptide binds to albumin with a K value of theinteraction that is at most 1×10⁻⁶ M, and even more preferably at most1×10⁻⁹ M (even tighter affinity). More preferably the K value of theinteraction that is at most 1×10⁻¹⁰ M, even more preferably is at most1×10⁻¹¹ M, yet even more preferably is at most 1×10⁻¹² M, and evenfurther is at most 1×10⁻¹³ M. For example, a K_(D) value of 1×10⁻¹⁴ M isa K value of the interaction that is at most 1×10⁻¹³ M. The K values canbe determined as described in PCT Published Appl. No. WO 2009/016043,preferably to human serum albumin. In one embodiment is contemplated theabove genus with the proviso that the amino acid sequence is notGVSDYYKNLINNAKTVEGVKALIDEI (SEQ ID NO:35).

As demonstrated herein and in the cited references, the albumin bindingcapacity of the ABD peptide can be retained despite amino acid changesso long as such changes retain sufficient tertiary structure of the ABDpeptide. Such changes include, for example, a substitution where anamino acid residue belonging to a certain functional grouping of aminoacid residues (e.g. hydrophobic, hydrophilic, polar etc.) is exchangedfor another amino acid residue from the same functional group.Accordingly, in one such embodiment of the ABD peptide, the motif X₅ isY. In one embodiment of the ABD X₈ is selected from N and R, and may inparticular be R. In one embodiment X₉ is L. In one embodiment X₁₁ isselected from N and S, and may in particular be N. In one embodiment X₁₂is selected from R and K, such as X₁₂ being R or X₁₂ being K. In oneembodiment X₁₄ is K. In one embodiment X₂₀ is selected from D, N, Q, E,H, S and R, and may in particular be E. In one embodiment X₂₃ isselected from K and I, and may in particular be K. In one embodiment X₂₄is selected from A, S, T, G, H and L. In a more specific embodiment X₂₄is L. In an even more specific embodiment “X₂₃ X₂₄” is KL. In anothereven more specific embodiment “X₂₃ X₂₄” is TL. In one embodiment X₂₄ isselected from A, S, T, G and H. In a more specific embodiment X₂₄ isselected from A, S, T, G and H and X₂₃ is I. In one embodiment X₂₅ is H.

The sequences of individual albumin binding motifs within the aboveformula include those presented as SEQ ID NOs:1-257 in PCT PublishedAppl. No. WO 2009/016043, incorporated herein by reference. In certainembodiments of the albumin binding polypeptide the albumin binding motifconsists of an amino acid sequence selected from SEQ ID NO:1-257. In amore specific embodiment of this aspect of the invention, the motifsequence is selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:9, SEQ IDNO:15, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:46, SEQ ID NO:49, SEQ IDNO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:155, SEQ ID NO:239, SEQ IDNO:240, SEQ ID NO:241, SEQ ID NO:242, SEQ ID NO:243, SEQ ID NO:244 andSEQ ID NO:245 of PCT Published Appl. No. WO 2009/016043. In yet morespecific embodiments of this aspect of the invention, the motif sequenceis selected from SEQ ID NO:3, SEQ ID NO:53 and SEQ ID NO:239 of PCTPublished Appl. No. WO 2009/016043. Albumin binding polypeptidescontaining albumin binding motifs, and thus suitable for conjugation orfusion to a hormone domain as described herein, are further describedherein and below and exemplified in Table 1 and the Examples. Not to bebound by theory but it is believed that the albumin binding motif canform part of a three-helix bundle protein domain. For example, the motifmay essentially constitute or form part of two alpha helices with aninterconnecting loop, within said three-helix bundle protein domain.Accordingly, in particular embodiments of the invention, such athree-helix bundle protein domain is selected from the group consistingof three-helix domains of bacterial receptor protein G fromStreptococcus strain G148. In different variants of this embodiment, thethree-helix bundle protein domain of which the motif forms a part isselected from the group consisting of domain GA1, domain GA2 and domainGA3 of protein G from Streptococcus strain G148, in particular domainGA3.

In embodiments of the present invention wherein the motif “forms part ofa three-helix bundle protein domain,” this is understood to mean thatthe sequence of the albumin binding motif is “inserted” into or“grafted” onto or “fused” to the sequence of the naturally occurring (orotherwise original) three-helix bundle domain, such that the motifreplaces a similar structural motif in the original domain. For exampleand without wishing to be bound by theory, the motif is thought toconstitute two of the three helices of a three-helix bundle, and canreplace such a two-helix motif within any three-helix bundle. Thereplacement of two helices of the three-helix bundle domain by the twomotif helices disclosed herein is performed so as not to affect thebasic structure of the polypeptide. That is, the overall folding of thebackbone of the polypeptide according to this embodiment of theinvention will be substantially the same as that of the three-helixbundle protein domain of which it forms a part, e.g. having the sameelements of secondary structure in the same order etc. Thus, a motifuseful to the engineered polypeptides herein can form part of athree-helix bundle domain if the polypeptide according to thisembodiment has the same fold as the original domain, implying that thebasic structural properties are shared, those properties e.g. resultingin similar CD spectra.

Accordingly, in one embodiment the albumin binding domain polypeptide isa three-helix bundle protein domain, which comprises the albumin bindingmotif as defined above and additional sequences making up the remainderof the three-helix configuration. To such an albumin binding domainpolypeptide can be fused to a leptin, a leptin analog, a leptin activefragment, or a leptin derivative thereof to create the engineeredpolypeptides as described herein. An albumin binding domain polypeptidesuitable for conjugation or fusion to a leptin, a leptin analog, aleptin active fragment, or a leptin derivative thereof can comprise theamino acid sequence:

(SEQ ID NO: 36)LAEAK X_(a) X_(b) A X_(c) X_(d) EL X_(e) KY -[ABM]- LAALP

wherein

[ABM] is an albumin binding motif as defined above, and,

independently of each other,

X_(a) is selected from V and E;

X_(b) is selected from L, E and D;

X_(c) is selected from N, L and I;

X_(d) is selected from R and K; and

X_(e) is selected from D and K.

In certain embodiments, X_(a) is E. In certain embodiments X_(b) is D.In certain embodiments, X_(c) is I. In certain embodiments, X_(d) is K.In certain embodiments, X_(a) independently is E, and/or independentlyX_(b) is D, and/or independently X_(c) is I, and/or independently X_(d)is K. In certain embodiments, the leucine at position 45 is present orabsent. In certain embodiments, the proline at position 46 is absent. Incertain embodiments, the albumin binding domain polypeptide isLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALTLHILAALP (SEQ ID NO: 50). Incertain embodiments, the albumin binding domain polypeptide isLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALISEILAALP (SEQ ID NO:51).

In a further embodiment, the ABD comprises one or more N-terminalhelix-capping amino acids, and in a further embodiment the helix-cappingamino acid may be serine, or may be glycine-serine. Accordingly for eachalbumin binding domain sequence disclosed herein, including those in thefigures and sequenced listing, also specifically contemplated for allaspects as disclosed herein in the engineered polypeptide, are albuminbinding domains, their Ser-ABD, Gly-Ser-ABD, Gly-ABD, Ala-ABD and theirdes-C-terminal-proline sequences.

Because of the presence of an albumin binding motif, the ABD peptidebinds to albumin with a K value of the interaction that is at most1×10⁻⁶ M and even more preferably at most 1×10⁻⁹ M (even tighteraffinity). More preferably the K value of the interaction that is atmost 1×10⁻¹⁰ M, even more preferably is at most 1×10⁻¹¹ M, yet even morepreferably is at most 1×10⁻¹² M, and even further is at most 1×10⁻¹³ M.

In one embodiment of this albumin binding polypeptide X_(a) is V. In oneembodiment of this polypeptide X_(b) is L. In one embodiment of thispolypeptide X_(c) is N. In one embodiment of this polypeptide X_(d) isR. In one embodiment of this polypeptide X_(e) is D.

Sequences of individual albumin binding domain polypeptides suitable forfusion with the active hormone domain peptides as described herein arepresented in Jonsson et al. (Id.) and as SEQ ID NOs:258-514 in PCTPublished Appl. No. WO 2009/016043, incorporated herein by reference.Selected compounds are disclosed in Table 1 below. Also encompassed bythe present invention is an albumin binding polypeptide having an aminoacid sequence with 85% or greater identity to a sequence selected fromSEQ ID NOs:258-514. In particular embodiments, the sequence of thealbumin binding polypeptide is selected from SEQ ID NO:259, SEQ IDNO:260, SEQ ID NO:266, SEQ ID NO:272, SEQ ID NO:282, SEQ ID NO:284, SEQID NO:303, SEQ ID NO:306, SEQ ID NO:310, SEQ ID NO:311, SEQ ID NO:312,SEQ ID NO:412, SEQ ID NO:496, SEQ ID NO:497, SEQ ID NO:498, SEQ IDNO:499, SEQ ID NO:500, SEQ ID NO:501 and SEQ ID NO:502 in PCT PublishedAppl. No. WO 2009/016043, and sequences having 85% or greater identitythereto. In yet further embodiments, the sequence of the albumin bindingpolypeptide is selected from SEQ ID NO:260, SEQ ID NO:270, SEQ IDNO:272, SEQ ID NO:291, SEQ ID NO:294, SEQ ID NO:298, SEQ ID NO:299, SEQID NO:300, SEQ ID NO:400, SEQ ID NO:484, SEQ ID NO:485, SEQ ID NO:486,SEQ ID NO:487, SEQ ID NO:488, SEQ ID NO:489 and SEQ ID NO:490 in PCTPublished Appl. No. WO 2009/016043, and sequences having 85% or greateridentity thereto. In still further embodiments, the sequence of thealbumin binding polypeptide is selected from SEQ ID NO:260, SEQ IDNO:310, SEQ ID NO:496, and SEQ ID NO: 511 in PCT Published Appl. No. WO2009/016043 and sequences having 85% or greater identity thereto.

In one embodiment, the albumin binding polypeptide further comprises oneor more additional amino acid residues positioned at the N- and/or theC-terminal of the sequence defined in SEQ ID NO:36. These additionalamino acid residues may play a role in enhancing the binding of albuminby the polypeptide, and improving the conformational stability of thefolded albumin binding domain, but may equally well serve otherpurposes, related for example to one or more of production,purification, stabilization in vivo or in vitro, coupling, labeling ordetection of the polypeptide, as well as any combination thereof. Suchadditional amino acid residues may comprise one or more amino acidresidue(s) added for purposes of chemical coupling, e.g. to an HD1.

The amino acids directly preceding or following the alpha helix at theN- or C-terminus of the amino acid sequence in SEQ ID NO:36 may thus inone embodiment affect the conformational stability. One example of anamino acid residue which may contribute to improved conformationalstability is a serine residue positioned at the N-terminal of SEQ IDNO:36 as defined above. The N-terminal serine residue may in some casesform a canonical S—X-X-E capping box, by involving hydrogen bondingbetween the gamma oxygen of the serine side chain and the polypeptidebackbone NH of the glutamic acid residue. This N-terminal capping maycontribute to stabilization of the first alpha helix of the three helixdomain constituting the albumin binding polypeptide according to thefirst aspect of the disclosure.

Thus, in one embodiment, the additional amino acids comprise at leastone serine residue at the N-terminal of the polypeptide. The amino acidsequence is in other words preceded by one or more serine residue(s). Inanother embodiment of the albumin binding polypeptide, the additionalamino acids comprise a glycine residue at the N-terminal of thepolypeptide. It is understood that the amino acid sequence of SEQ IDNO:36 may be preceded by one, two, three, four or any suitable number ofamino acid residues. Thus, the amino acid sequence may be preceded by asingle serine residue, a single glycine residue or a combination of thetwo, such as a glycine-serine (GS) combination or aglycine-serine-serine (GSS) combination. In yet another embodiment, theadditional amino acid residues comprise a glutamic acid at theN-terminal of the polypeptide as defined by the sequence of SEQ IDNO:36.

Exemplary ABD species include, but are not limited to, the compounds setforth in Table 1 following and the Examples. See also PCT PublishedAppl. No. WO 2009/016043, incorporated herein by reference in itsentirety and for all purposes. An ABD peptide useful in compounds,methods and pharmaceuticals compositions described herein can be afragment or analog of an ABD peptide disclosed herein or known in theart so long as it contains an albumin binding motif and binds albuminwith the affinity described herein.

TABLE 1 Selected ABD peptides ABD peptide sequence SEQ ID NO:LAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP 37LAEAKVLANRELDKYGVSDFYKSYINRAKTVEGVHTLIGHILAALP 38LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVNALTHHILAALP 39LAEAKVLANRELDKYGVSDYYKNLINRARTVEGVHALIDHILAALP 40LAEAKVLANRELDKYGVSDYYKNIINRAKTVEGVRALKLHILAALP 41LAEAKVLANRELDKYGVSDFYKNLINRAKTVEGVSSLKGHILAALP 42LAEAKVLANRELDKYGVSDYYKNLINKAKTVEGVEALTLHILAALP 43LAEAKVLANRELDKYGVSDFYKNLINRAKTVEGVDALIAHILAALP 44LAEAKVLANRELDKYGVSDFYKSLINRAKTVEGVDALTSHILAALP 45LAEAKVLANRELDKYGVSDFYKNLINRAKTVEGVNSLTSHILAALP 46LAEAKVLANRELDKYGVSDFYKNVINKAKTVEGVEALIADILAALP 47LAEAKVLANRELDKYGVSDYYKNLINKAKTVEGVQALIAHILAALP 48LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP 49LAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALTLHILAALP 50LAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALISEILAALP 51LAEAKEDAIKELDKYGVSDYYKRLISKAKTVEGVKALISEILAALP 52

Binding to Albumin.

Serum albumin is the most abundant protein in mammalian sera (40 g/L;approximately 0.7 mM in humans) where it binds a variety of moleculesincluding but not limited to lipids and bilirubin (Peters T, 1985,Advances in Protein Chemistry 37:161). It has been observed that thehalf-life of serum albumin is directly proportional to the size of theanimal, where for example human serum albumin (HSA) has a half-life of19 days and rabbit serum albumin has a half-life of about 5 days(McCurdy T R et al., J. Lab. Clin. Med. 143:115, 2004). Human serumalbumin is widely distributed throughout the body, in particular in theintestinal and blood compartments, where it is mainly involved in themaintenance of osmolarity. Structurally, albumins are single-chainproteins comprising three homologous domains and totaling 584 or 585amino acids (Dugaiczyk L et al., 1982, Proc. Natl. Acad. Sci. USA79:71). Albumins contain 17 disulfide bridges and a single reactivethiol, C34, but lack N-linked and O-linked carbohydrate moieties(Peters, 1985, Id.; Nicholson J P et al., 2000, Br J Anaesth 85:599).The lack of glycosylation simplifies recombinant expression of albumin.This property of albumin, together with the fact that itsthree-dimensional structure is known (see e.g., He X M & Carter D C,1992, Nature 358:209), has made it an attractive candidate for use inrecombinant fusion proteins. Such fusion proteins generally combine atherapeutic protein (which would be rapidly cleared from the body uponadministration of the protein per se) and a plasma protein (whichexhibits a natural slow clearance) in a single polypeptide chain. Seee.g., Sheffield WP, 2001, Curr. Drug Targets Cardiovacs. Haematol.Disord. 1:1). Such proteins may provide clinical benefits in requiringless frequent injection and higher levels of therapeutic protein invivo. However, the engineered polypeptides herein are not conjugated toalbumin, but instead contain motifs that allow non-covalent binding toalbumin.

Further Embodiments

It is understood that each of the polypeptides disclosed herein are alsocontemplated to include (optionally) a methionine at the N-terminus inframe with the naturally-occurring first amino acid thereof. Forexample, metreleptin (leptin A100) consists of mature human leptin towhich has been added an N-terminal methionine, as disclosed in SEQ IDNO:20. Similarly, a methionine residue may be included at the N-terminusof any of the amino acid sequences and Formulae disclosed hereinthroughout. It is further understood that where a C-terminal Gly appearsin an engineered polypeptide sequence set forth herein, the residue maybe lost during subsequent amidation.

In some embodiments, a leptin, a leptin analog, a leptin activefragment, or a leptin derivative can have at least 50%, for example 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or even higher,sequence identity relative to a parent leptin. In some embodiments, theparent leptin is a leptin set out in SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, IDNO:143, SEQ ID NO:144, SEQ ID NO:145, or SEQ ID NO:146. Accordingly, insome embodiments, a leptin, a leptin analog, a leptin active fragment,or a leptin derivative may have at least 50%, for example 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or even higher, sequence identityrelative to any leptin selected from the group consisting of SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQID NO:23. In some embodiments, a leptin, a leptin analog, a leptinactive fragment, or a leptin derivative may have at least 50%, forexample 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or evenhigher, sequence identity relative to the leptin set forth in SEQ IDNO:20. In some embodiments, a leptin, a leptin analog, a leptin activefragment, or a leptin derivative may have at least 50%, for example 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or even higher,sequence identity relative to any leptin selected from the groupconsisting SEQ ID NO:24, SEQ ID NO: 25, SEQ ID NO:26, SEQ ID NO:27, SEQID NO: 28, or SEQ ID NO:29. In some embodiments, a leptin analog mayhave at least 90% sequence identity relative to the leptin set forth inSEQ ID NO:20. In some embodiments, a leptin analog may have at least 50%sequence identity relative to the leptin set forth in SEQ ID NO:1, SEQID NO:2, ID NO:143, SEQ ID NO:144, SEQ ID NO:145, or SEQ ID NO:146. Insome embodiments, a leptin analog may have at least 90% sequenceidentity relative to the leptin set forth in SEQ ID NO:1, SEQ ID NO: 2,ID NO:143, SEQ ID NO:144, SEQ ID NO:145, or SEQ ID NO:146. In someembodiments, a leptin analog may have at least 50% sequence identityrelative to the leptin set forth in SEQ ID NO:14 or SEQ ID NO:15. Insome embodiments, a leptin analog may have at least 90% sequenceidentity relative to the leptin set forth in SEQ ID NO:14 or SEQ IDNO:15. In some embodiments, a leptin analog may have at least 50%sequence identity relative to the leptin set forth in SEQ ID NO: 32. Insome embodiments, a leptin analog may have at least 90% sequenceidentity relative to the leptin set forth in SEQ ID NO:32. In someembodiments, a leptin analog may have at least 50% sequence identityrelative to the leptin set forth in SEQ ID NO: 33. In some embodiments,a leptin analog may have at least 90% sequence identity relative to theleptin set forth in SEQ ID NO:33. In some embodiments, a leptin analogmay have at least 50% sequence identity relative to the leptin set forthin SEQ ID NO:10 or SEQ ID NO:11. In some embodiments, a leptin analogmay have at least 90% sequence identity relative to the leptin set forthin SEQ ID NO:10 or SEQ ID NO:11. In some embodiments, a leptin analogmay have at least 50% sequence identity relative to the leptin set forthin SEQ ID NO:12 or SEQ ID NO:13. In some embodiments, a leptin analogmay have at least 90% sequence identity relative to the leptin set forthin SEQ ID NO:12 or SEQ ID NO:13. Additionally, leptins may be designed,prepared, and used in accordance with the invention in which 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or even 21amino acids of a leptin selected from the group consisting of: SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, andSEQ ID NO:146; is/are substituted with another amino acid, such as aconservative amino acid or a non-conservative amino acid, or is/areotherwise altered. As customary in the art, the term “conservative” inthe context of amino acid substitutions refers to substitution whichmaintains properties of charge type (e.g., anionic, cationic, neutral,polar and the like), hydrophobicity or hydrophilicity, bulk (e.g., vander Waals contacts and the like), and/or functionality (e.g., hydroxy,amine, sulhydryl and the like). The term “non-conservative” refers to anamino acid substitution which is not conservative.

Additionally, as is understood in the art, for example, murine leptins,rat leptins, bovine leptins, porcine leptins, and rhesus monkey leptins,such as those disclosed herein, are each substantially homologous tohuman leptins; in particular, the mature forms of these leptins aresubstantially homologous to mature leptins, and further, particularlynear the N-terminal portion of the protein. One may prepare analogs ofsuch leptins, such as mature human leptin form 1 (SEQ ID NO:16) andmetreleptin (SEQ ID NO:20), such as by substituting or otherwisealtering amino acid residues at one or more positions in such sequenceswhere divergence is observed in a corresponding mature mouse, rat,bovine, porcine, or rhesus monkey leptin. For example, mature humanleptins (e.g., SEQ ID NO:20) elicits biological responses in, forexample, mice, rat, and monkey). See, e.g., WO 98/28427, WO 2009/064298,US2007/0020284, US2008/0207512, and Murakami et al., 1995, Biochem.Biophys. Res. Comm. 209: 944-952. Because human mature leptins havebiological activity in, e.g., such species, leptins may be designed andprepared in which one or more amino acids at positions which aredivergent at the corresponding position(s) in a leptin from one or moreof such species are substituted with the amino acid(s) at suchcorresponding divergent positions.

For example, using a human mature leptin protein according to SEQ IDNO:16 wherein

the first amino acid is valine and the amino acid at position 146 iscysteine, one may substitute with another amino acid one or more of theamino acids at positions 32, 35, 50, 64, 68, 71, 74, 77, 89, 97, 100,101, 105, 106, 107, 108, 111, 118, 136, 138, 142, and 145 with thecorresponding amino acid(s) found at the corresponding position(s) inSEQ ID NO:143) in order to design, prepare, and use engineeredpolypeptides in accordance with the invention. Additionally, one mayalso substitute another amino acid, such as a conservative amino acid ora non-conservative amino acid, into one or more of positions 32, 35, 50,64, 68, 71, 74, 77, 89, 97, 100, 101, 105, 106, 107, 108, 111, 118, 136,138, 142, and 145 of, for example, SEQ ID NO:16 in order to design,prepare, and use engineered polypeptides in accordance with theinvention.

One may further prepare additional leptins based on the mature ratleptin protein sequence (SEQ ID NO:12). See, e.g., WO 98/28427,US2007/0020284, and Murakami et al., 1995, Id., herein incorporated byreference in their entireties and for all purposes. Mature rat leptindiffers from mature human leptin form 1 (SEQ ID NO:16) at the followingpositions: 4, 32, 33, 35, 50, 68, 71, 74, 77, 78, 89, 97, 100, 101, 102,105, 106, 107, 108, 111, 118, 136, 138 and 145. Accordingly, at one ormore of such positions in SEQ ID NO:16, one may substitute the aminoacid found at the corresponding position(s) found in mature rat leptin(SEQ ID NO:12) in order to design, prepare, and use engineeredpolypeptides in accordance with the invention. Additionally, one mayalso substitute another amino acid, such as a conservative amino acid ora non-conservative amino acid, into one or more of positions 4, 32, 33,35, 50, 68, 71, 74, 77, 78, 89, 97, 100, 101, 102, 105, 106, 107, 108,111, 118, 136, 138 and 145 of, for example, SEQ ID NO:16, in order todesign, prepare, and use engineered polypeptides in accordance with theinvention.

The positions from both mature rat leptin (SEQ ID NO:12) and maturemurine leptin form 1 (SEQ ID NO:143) which diverge from the mature humanleptin form 1 (SEQ ID NO:16) are: 4, 32, 33, 35, 50, 64, 68, 71, 74, 77,78, 89, 97, 100, 102, 105, 106, 107, 108, 111, 118, 136, 138, 142, and145. Accordingly, at one or more of such positions in SEQ ID NO:16, onemay substitute the amino acid found at the corresponding position(s)found in mature rat leptin sequence (SEQ ID NO:12) or mature murine form1 sequence (SEQ ID NO:143) in order to design, prepare, and useengineered polypeptides in accordance with the invention. Additionally,one may also substitute another amino acid, such as a conservative aminoacid or a non-conservative amino acid, into one or more of positions 4,32, 33, 35, 50, 64, 68, 71, 74, 77, 78, 89, 97, 100, 102, 105, 106, 107,108, 111, 118, 136, 138, 142, and 145 in order to design, prepare, anduse engineered polypeptides in accordance with the invention.

In addition, the amino acids found in rhesus monkey mature leptin (SEQID NO:10) which diverge from mature human leptin form 1 (SEQ ID NO:16)are (with amino acid residues noted in parentheses in one letter aminoacid abbreviation): 8 (S), 35 (R), 48(V), 53(Q), 60(I), 66(I), 67(N),68((L), 89(L), 100(L), 108(E), 112 (D), and 118 (L). Since human matureleptins elicit biological response monkeys, a leptin, such as maturehuman leptin form 1 (SEQ ID NO:16) having one or more of the rhesusmonkey divergent amino acids replaced with another amino acid, such asthe amino acids in parentheses, may be employed in designing, preparing,and using engineered polypeptides in accordance with the invention. Itshould be noted that certain rhesus divergent amino acids are also thosefound in, for example, the above mature murine leptin form 1 (positions35, 68, 89, 100 and 112). Thus, one may prepare leptins in which one ormore amino acids at positions 4, 8, 32, 33, 35, 48, 50, 53, 60, 64, 66,67, 68, 71, 74, 77, 78, 89, 97, 100, 102, 105, 106, 107, 108, 111, 112,118, 136, 138, 142, and 145 of, e.g., mature human leptin form 1 (SEQ IDNO:16) are replaced by the corresponding amino acid(s) at suchposition(s) in murine or rhesus monkey leptins (e.g., SEQ ID NO:143and/or SEQ ID NO:10).

Other leptins may be prepared by deleting a part of a leptin amino acidsequence, provided that such a leptin amino acid sequence may elicit abiological response. Such leptin amino acid sequences are leptin activefragments. For example, mature murine leptins, mature rhesus monkeyleptins, mature human leptins, and mature rat leptins, and other leptinsall lack the N-terminal 21 amino acid signal sequence that is present inthe unprocessed, full-length forms of such leptin.

One may prepare the following active leptin fragments of such matureleptins:

(a) amino acids 98-146

(b) amino acids 1-32

(c) amino acids 40-116

(d) amino acids 1-99 and (connected to) 112-146

(e) amino acids 1-99 and (connected to) 112-146 having one or more ofamino acids 100-111 placed between amino acids 99 and 112.

In addition, such active leptin fragments may also be prepared in whichone or more of the amino acids at positions in, e.g., mature humanleptin form 1 that are substituted with the amino acids found at thecorresponding position(s) found in, e.g., rat, murine, monkey, porcine,and/or bovine mature leptins as disclosed above. Furthermore, anysubstitutions or alterations may be in the form of altered amino acids,such as peptidomimetics or D-amino acids.

Additionally, the present invention encompasses engineered polypeptideswhich comprise a leptin, a leptin analog, a leptin active fragment, or aleptin derivative as described above, wherein the a leptin, a leptinanalog, a leptin active fragment, or a leptin derivative is selectedfrom:

(a) the amino acid sequence 1-146 of a leptin selected from the groupconsisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQID NO:8, SEQ ID NO:10, SEQ ID NO:12. SEQ ID NO:13, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:143, and SEQ ID NO:144; inwhich a different amino acid is substituted in one or more of thefollowing positions and retaining the same numbering (even in theabsence of a glutaminyl residue at position 28): 4, 32, 33, 35, 50, 64,68, 71, 74, 77, 78, 89, 97, 100, 102, 105, 106, 107, 108, 111, 118, 136,138, 142, and 145;

(b) the amino acid sequence of subpart (a) in which the glutaminylresidue at position 28 is absent;

(c) the amino acid sequence of subparts (a) or (b) in which a methionylresidue is added at the N-terminus;

(d) a leptin consisting of a fragment of the amino acid sequence of (a),(b), or (c) selected from the group consisting of:

-   -   (i) amino acids 98-146    -   (ii) amino acids 1-32    -   (iii) amino acids 40-116    -   (iv) amino acids 1-99 and 112-146    -   (v) amino acids 1-99 and 112-146 in which one or more of amino        acids 100-111 is placed between amino acids 99 and 112; and,    -   (vi) the amino acid sequence of subpart (i) wherein one or more        of amino acids 100, 102, 105, 106, 107, 108, 111, 118, 136, 138,        142, and 145 is substituted with another amino acid;    -   (vii) the amino acid sequence of subpart (ii) wherein one or        more of amino acids 4, 8 and 32 is substituted with another        amino acid;    -   (viii) the amino acid sequence of subpart (iii) wherein one or        more of amino acids 50, 53, 60, 64, 66, 67, 68, 71, 74, 77, 78,        89, 97, 100, 102, 105, 106, 107, 108, 111 and 112 is replaced        with another amino acid;    -   (ix) the amino acid sequence of subpart (iv) wherein one or more        of amino acids 4, 8, 32, 33, 35, 48, 50, 53, 60, 64, 66, 67, 68,        71, 74, 77, 78, 89, 97, 112, 118, 136, 138, 142, and 145 is        replaced with another amino acid; and    -   (x) the amino acid sequence of subpart (v) wherein one or more        of amino acids 4, 32, 33, 35, 50, 64, 68, 71, 74, 77, 78, 89,        97, 100, 102, 105, 106, 107, 108, 111, 118, 136, 138, 142, and        145 is replaced with another amino acid;    -   (xi) the leptin of any of subparts (i)-(x) wherein a methionine        has been added at the N-terminus; and

(e) the leptin of any of subparts (a) through (e) to which a chemicalmoiety is attached;

(f) the leptin of subpart (g) wherein said chemical moiety is a watersoluble polymer moiety;

(g) a leptin of subpart (f) wherein said water soluble polymer moiety ispolyethylene glycol;

(h) a leptin of subpart (f) wherein said water soluble polymer moiety isa polyaminoacid moiety; and

(i) a leptin of any one of subparts (e) through (h) wherein said moietyis attached at solely the N-terminus of said protein moiety.

With regard to the above, leptins to which a chemical moiety is attachedare leptin derivatives. Derivatization of leptins by attachment of oneor more chemical moieties has been found to provide some advantage undercertain circumstances, such as increasing the stability and circulationtime of the therapeutic protein and decreasing immunogenicity andpropensity for, for example, generation of neutralizing antibodiesand/or incidence of injection site reactions. See, e.g., WO 98/28427,US2007/0020284, U.S. Pat. No. 4,179,337, Davis et al., issued Dec. 18,1979. For a review, see Abuchowski et al., in ENZYMES AS DRUGS. (J. S.Holcerberg and J. Roberts, eds. pp. 367-383 (1981)); Francis et al., Id.Accordingly, when employing a derivatized leptin and an ABM or an ABD,one may advantageously generate engineered polypeptides of the inventionpossessing advantages provided by both entities.

Leptin derivatives may constitute leptins to which a chemicalmodification has been made of one or more of its amino acid side groups,α-carbon atoms, terminal amino group, or terminal carboxylic acid group.A chemical modification includes, but is not limited to, attaching oneor more chemical moieties, creating new bonds, and removing one or morechemical moieties. Modifications at amino acid side groups include,without limitation, alkylation, acylation, ester formation, amideformation, maleimide coupling, acylation of lysine ε-amino groups,N-alkylation of arginine, histidine, or lysine, alkylation of glutamicor aspartic carboxylic acid groups, and deamidation of glutamine orasparagine. Modifications of the terminal amino include, withoutlimitation, the desamino, N-lower alkyl, N-di-lower alkyl, and N-acylmodifications. Modifications of the terminal amino include, withoutlimitation, the desamino, N-lower alkyl, N-di-lower alkyl, and N-acylmodifications, such as alkylacyls, branched alkylacyls, alkylaryl-acyls.Modifications of the terminal carboxy group include, without limitation,the amide, lower alkyl amide, dialkyl amide, arylamide, alkylarylamideand lower alkyl ester modifications. Lower alkyl is C₁-C₄ alkyl.Furthermore, one or more side groups, or terminal groups, may beprotected by protective groups known to the ordinarily-skilled syntheticchemist. The α-carbon of an amino acid may be mono- or dimethylated.

Such derivatives include leptins conjugated to one or more water solublepolymer molecules, such as polyethylene glycol (“PEG”) or fatty acidchains of various lengths (e.g., stearyl, palmitoyl, octanoyl), by theaddition of polyamino acids, such as poly-his, poly-arg, poly-lys, andpoly-ala, or by addition of small molecule substituents include shortalkyls and constrained alkyls (e.g., branched, cyclic, fused,adamantyl), and aromatic groups. In some embodiments, the water solublepolymer molecules will have a molecular weight ranging from about 500Daltons to about 60,000 Daltons.

Such polymer-conjugations may occur singularly at the N- or C-terminusor at the side chains of amino acid residues within the sequence of aleptin as disclosed herein. Alternatively, there may be multiple sitesof derivatization along the amino acid sequence of such a leptin.Substitution of one or more amino acids with lysine, aspartic acid,glutamic acid, or cysteine may provide additional sites forderivatization. See, e.g., U.S. Pat. Nos. 5,824,784 and 5,824,778. Insome embodiments, a leptin may be conjugated to one, two, or threepolymer molecules.

In some embodiments, the water soluble polymer molecules are linked toan amino, carboxyl, or thiol group, and may be linked by N or C termini,or at the side chains of lysine, aspartic acid, glutamic acid, orcysteine. Alternatively, the water soluble polymer molecules may belinked with diamine and dicarboxylic groups. In some embodiments, aleptin is conjugated to one, two, or three PEG molecules through anepsilon amino group on a lysine amino acid.

Leptin derivatives also include leptins with chemical alterations to oneor more amino acid residues. Such chemical alterations includeamidation, glycosylation, acylation, sulfation, phosphorylation,acetylation, and cyclization. The chemical alterations may occursingularly at the N- or C-terminus or at the side chains of amino acidresidues within the sequence of a leptin. In one embodiment, theC-terminus of these peptides may have a free —OH or —NH₂ group. Inanother embodiment, the N-terminal end may be capped with anisobutyloxycarbonyl group, an isopropyloxycarbonyl group, ann-butyloxycarbonyl group, an ethoxycarbonyl group, an isocaproyl group(“isocap”), an octanyl group, an octyl glycine group (denoted as“G(Oct)” or “octylGly”), an 8-aminooctanic acid group, a dansyl, and/ora Fmoc group. In some embodiments, cyclization can be through theformation of disulfide bridges. Alternatively, there may be multiplesites of chemical alteration along the leptin amino acid sequence.

In certain embodiments, leptins are chemically altered to include aBolton-Hunter group. Bolton-Hunter reagents are known in the art(“Radioimmunoassay and related methods,” A. E. Bolton and W. M. Hunter,Chapter 26 of HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, VOLUME I,IMMUNOCHEMISTRY, edited by D. M. Weir, Blackwell ScientificPublications, 1986), and may be used to introduce tyrosine-like moietieswith a neutral linkage, through amino-terminal α-amino groups or ε-aminogroups of lysine. In some embodiments, the N-terminal end of a leptin ismodified with a Bolton-Hunter group. In some embodiments, an internallysine residue is modified with a Bolton-Hunter group. In someembodiments, there may be multiple sites of Bolton-Hunter modificationalong the leptin amino acid sequence. Bolton-Hunter reagents used forpolypeptide modification are commercially available, and may include,but are not limited to, water-soluble Bolton-Hunter reagent,Sulfosuccinimidyl-3-[4-hydrophenyl]propionate (Pierce Biotechnology,Inc., Rockford, Ill.) and Bolton-Hunter reagent-2, N-Succinimidyl3-(4-hydroxy-3-iodophenyl) Priopionate (Wako Pure Chemical Industries,Ltd., Japan, catalog #199-09341). An exemplary Bolton-Hunter groupconjugated through an amide linkage to a leptin is illustrated below,wherein the dashed line passes through the amide bond:

Leptins may be iodinated (such as radiolabeled with ¹²⁵I) before orafter Bolton-Hunter modification.

In order to prepare engineered polypeptides in accordance with theinvention, a leptin derivative for use in the preparation of such mayinclude one or more modifications of a “non-essential” amino acidresidue. In the context of the invention, a “non-essential” amino acidresidue is a residue that can be altered, e.g., derivatized, withoutabolishing or substantially reducing the activity (e.g., the agonistactivity) of the leptin. The engineered polypeptides of the inventionmay include derivatizations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moreamino acid residues of the leptin moiety; of these, one or more aminoacid residues may be non-essential amino acid residues. Additionally,the polypeptides of the invention may be derivatized such that theyinclude additions of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moreamino acids of the leptin moiety without abolishing or substantiallyreducing the activity of the polypeptide. Additionally, suchnon-essential amino acid residues may be substituted with an amino acidresidue that is amenable to derivatization as described throughout.

As used throughout, “amino acid,” “amino acid residue” and the likerefer to natural amino acids, unnatural amino acids, and modified aminoacids. Unless stated to the contrary, any reference to an amino acid,generally or specifically by name, includes reference to both the D andthe L stereoisomers if their structure allow such stereoisomeric forms.Natural amino acids include alanine (Ala), arginine (Arg), asparagine(Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamicacid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine(Leu), Lysine (Lys), methionine (Met), phenylalanine (Phe), proline(Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr)and valine (Val). Unnatural amino acids include, but are not limited tohomolysine, homoarginine, homoserine, azetidinecarboxylic acid,2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, aminopropionicacid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid,2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisbutyric acid,2-aminopimelic acid, tertiary-butylglycine, 2,4-diaminoisobutyric acid,desmosine, 2,2′-diaminopimelic acid, 2,3-diaminopropionic acid,N-ethylglycine, N-ethylasparagine, homoproline, hydroxylysine,allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine,allo-isoleucine, N-methylalanine, N-methylglycine, N-methylisoleucine,N-methylpentylglycine, N-methylvaline, naphthalanine, norvaline,norleucine, ornithine, pentylglycine, pipecolic acid and thioproline.Additional unnatural amino acids include modified amino acid residueswhich are chemically blocked, reversibly or irreversibly, or chemicallymodified on their N-terminal amino group or their side chain groups, asfor example, N-methylated D and L amino acids or residues wherein theside chain functional groups are chemically modified to anotherfunctional group. For example, modified amino acids include methioninesulfoxide; methionine sulfone; aspartic acid-(beta-methyl ester), amodified amino acid of aspartic acid; N-ethylglycine, a modified aminoacid of glycine; or alanine carboxamide, a modified amino acid ofalanine. Additional residues that can be incorporated are described inSandberg et al., J. Med. Chem. 41: 2481-91, 1998.

As mentioned above, chemical moieties suitable for such derivatizationof leptins and other polypeptides include, for example, various watersoluble polymers. Preferably, for therapeutic use of the end-productpreparation, the polymer will be pharmaceutically acceptable. Oneskilled in the art will be able to select the desired polymer based onsuch considerations as whether the polymer/protein conjugate will beused therapeutically, and if so, the desired dosage, circulation time,resistance to proteolysis, and other considerations. For the engineeredpolypeptides and leptins, the effectiveness of the derivatization may beascertained by administering the derivatized leptin or the derivatizedengineered polypeptide, in the desired form (i.e., by osmotic pump, or,more preferably, by injection or infusion, or, further formulated fororal, pulmonary or nasal delivery, for example), and observingbiological effects and biological responses as described herein.

Such a water soluble polymer may be selected from the group consistingof, for example, polyethylene glycol, copolymers of ethyleneglycol/propylene glycol, carboxymethylcellulose, dextran, polyvinylalcohol, polyvinyl pyrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymersor random copolymers), and dextran or poly(n-vinylpyrolidone)polyethylene glycol, propylene glycol homopolymers,polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyolsand polyvinyl alcohol. Polyethylene glycol propionaldehyde may haveadvantages in manufacturing due to its stability in water. Also,succinate and styrene may also be used.

Leptin derivatives used in the design and preparation of engineeredpolypeptides in accordance with the invention may be prepared byattaching polyaminoacids or branch point amino acids to the leptinmoiety. For example, the polyaminoacid may be an additional carrierprotein, such as an Fc moiety, which can serve to also increase thecirculation half life of the leptin or the engineered polypeptide, inaddition to the advantages achieved via attachment of an ABM or an ABD.Additionally, such polyaminoacids may be selected from the groupconsisting of serum album (such as human serum albumin), an additionalantibody or portion thereof (e.g. the Fc region), or otherpolyaminoacids, e.g. polylysines. As indicated below, the location ofattachment of the polyaminoacid may be at the N-terminus of the leptinmoiety, or C-terminus, or other places in between, and also may beconnected by a chemical “linker” moiety to the leptin, such as apeptidic linker or a non-peptidic linker.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 2 kilodaltons (kDa) and about 100 kDa (the term “about”indicating that in preparations of polyethylene glycol, some moleculeswill weigh more, some less, than the stated molecular weight) for easein handling and manufacturing. In certain embodiments, the polyethyleneglycol is between about 2 kDa and about 60 kDa. In certain embodiments,the polyethylene glycol is between about 2 kDa and about 40 kDa. Incertain embodiments, the polyethylene glycol is between about 5 kDa andabout 40 kDa. In certain embodiments, the polyethylene glycol is betweenabout 10 kDa and about 40 kDa. In certain embodiments, the polyethyleneglycol is between about 5 kDa and about 30 kDa. In certain embodiments,the polyethylene glycol is between about 5 kDa and about 20 kDa. Incertain embodiments, the polyethylene glycol is between about 10 kDa andabout 20 kDa. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,solubility characteristics, the effects, if any, on biological activity,the ease in handling, the degree or lack of antigenicity and other knowneffects of the polyethylene glycol attached to a leptin and/or to anengineered polypeptide of the invention). Additional considerations thatmay influence the selection of a PEG of a particular molecular weightwhich may be attached to a leptin to generate a leptin derivative inaccordance with the invention include the extent to which such amolecular weight PEG may: mitigate aggregation and/or increase thesolubility of the leptin and/or the engineered polypeptide, when presentin a pharmaceutically acceptable composition or formulation, or whenexposed to physiological fluids or tissues upon administration to asubject (such as by injection); mitigate the incidence of injection sitereactions caused by administration of the leptin or the engineeredpolypeptide upon administration to a subject by injection; mitigate thegeneration of neutralizing antibodies that may be raised against theleptin or the engineered polypeptide as a result of administration ofsuch a leptin or an engineered polypeptide to a subject; and the like.

The number of polymer molecules so attached may vary, and one skilled inthe art will be able to ascertain the resultant effect on function. Onemay mono-derivatize, or may provide for a di-, tri-, tetra- or somecombination of derivatization, with the same or different chemicalmoieties (e.g., polymers, such as different weights of polyethyleneglycols). The proportion of polymer molecules to leptin molecules orengineered polypeptide molecules to be derivatized will vary, as willtheir concentrations in the reaction mixture. In general, the optimumratio, in terms of efficiency of reaction in that there is no excessunreacted leptin (or engineered polypeptide, as the case may be) orpolymer, will be determined by factors such as the desired degree ofderivatization (e.g., mono, di-, tri-, etc.), the molecular weight ofthe polymer selected, whether the polymer is branched or unbranched, andthe reaction conditions.

The chemical moieties should be attached to the leptin and/or theengineered polypeptide with consideration of the effects on functionalor antigenic domains of the leptin and/or to the engineered polypeptide.There are a number of attachment methods available to those skilled inthe art. E.g., EP 0 401 384 herein incorporated by reference (couplingPEG to G-CSF), see also Malik et al., 1992, Exp. Hematol. 20:1028-1035(reporting pegylation of GM-CSF using tresyl chloride). For example,polyethylene glycol may be covalently bound through amino acid residuesvia a reactive group, such as, a free amino or carboxyl group. Reactivegroups are those to which an activated polyethylene glycol molecule maybe bound. The amino acid residues having a free amino group may includelysine residues and the N-terminal amino acid residue. Those having afree carboxyl group may include aspartic acid residues, glutamic acidresidues, and the C-terminal amino acid residue. Sulfhydryl groups mayalso be used as a reactive group for attaching the polyethylene glycolmolecule(s). Preferred for therapeutic purposes is attachment at anamino group, such as attachment at the N-terminus or lysine group.Attachment at residues important for receptor binding should be avoidedif receptor binding is desired.

One may specifically desire to design and prepare an N-terminallychemically modified leptin for use in the preparation of engineeredpolypeptides of the invention. Using polyethylene glycol as anillustration of the present compositions, one may select from a varietyof polyethylene glycol molecules (by molecular weight, branching, etc.),the proportion of polyethylene glycol molecules to leptin or engineeredpolypeptide molecules, as the case may be, in the reaction mix, the typeof pegylation reaction to be performed, and the method of obtaining theselected N-terminally pegylated protein. The method of obtaining theN-terminally pegylated preparation (i.e., separating this moiety fromother monopegylated moieties if necessary) may be by purification of theN-terminally pegylated material from a population of pegylated proteinmolecules. Selective N-terminal chemical modification may beaccomplished by reductive alkylation which exploits differentialreactivity of different types of primary amino groups (lysine versus theN-terminal) available for derivatization in a particular protein. Underthe appropriate reaction conditions, substantially selectivederivatization of the protein at the N-terminus with a carbonyl groupcontaining polymer is achieved. For example, one may selectivelyN-terminally pegylate the protein by performing the reaction at a pHwhich allows one to take advantage of the pK_(a) differences between theε-amino group of the lysine residues and that of the α-amino group ofthe N-terminal residue of the protein. By such selective derivatization,attachment of a water soluble polymer to a protein is controlled: theconjugation with the polymer takes place predominantly at the N-terminusof the protein and no significant modification of other reactive groups,such as the lysine side chain amino groups, occurs. Using reductivealkylation, the water soluble polymer may be of the type describedabove, and should have a single reactive aldehyde for coupling to theprotein. Polyethylene glycol propionaldehyde, containing a singlereactive aldehyde, may be used.

In some embodiments, compounds are provided having a linker, for exampleL1, as described herein, covalently linking a polypeptide hormone domainwith an ABD peptide. In some embodiments, a first linker (L1) covalentlylinks HD1 within the engineered polypeptide. In some embodiments, thepolypeptide hormone domain (e.g., HD1) as described herein) can becovalently linked to the ABD peptide via a peptide linker. Any linker isoptional; i.e., any linker may simply be a bond. When present thechemical structure of a linker is not critical because it serves mainlya spacer function. In one embodiment the linker comprises from 1 to 30or less amino acids linked by peptide bonds. The amino acids can beselected from the 20 naturally occurring amino acids. Alternatively,non-natural amino acids can be incorporated either by chemicalsynthesis, post-translational chemical modification or by in vivoincorporation by recombinant expression in a host cell. Some of theseamino acids may be glycosylated.

In certain embodiments the 1 to 30 or less amino acids are selected fromglycine, alanine, proline, asparagine, glutamine, lysine, aspartate, andglutamate. In a further embodiment the linker is made up of a majorityof amino acids that are sterically unhindered, such as glycine, alanineand/or serine. Polyglycines are particularly useful, e.g. (Gly)₃, (Gly)₄(SEQ ID NO:116), (Gly)₅ (SEQ ID NO:117), as are polyalanines,poly(Gly-Ala), poly(Glyn-Ser), poly (Gly_(n)-Glu), poly(Gly_(n)-Lys),poly(Gly_(n)-Asp), and poly(Gly_(n)-Arg) motifs. Other specific examplesof linkers are (Gly)₃Lys(Gly)₄ (SEQ ID NO:118); (Gly)₃AsnGlySer(Gly)₂(SEQ ID NO:119); (Gly)₃Cys(Gly)₄ (SEQ ID NO:120); and GlyProAsnGlyGly(SEQ ID NO:121). Combinations of Gly and Ala are particularly useful asare combination of Gly and Ser. Thus, in a further embodiment thepeptide linker is selected from the group consisting of a glycine richpeptide, e.g. Gly-Gly-Gly; the sequences [Gly-Ser]_(n) (SEQ ID NO:122),[Gly-Gly-Ser]_(n) (SEQ ID NO:123), [Gly-Gly-Gly-Ser]_(n) (SEQ ID NO:124)and [Gly-Gly-Gly-Gly-Ser]_(n) (SEQ ID NO:125), where n is 1, 2, 3, 4, 5,6, 7, 8, 9, or 10, for example, [Gly-Gly-Gly Ser]₁ (SEQ ID NO: 149),[Gly-Gly-Gly-Gly Ser]₁ (SEQ ID NO: 150), [Gly-Gly-Gly Ser]₄ (SEQ ID NO:151), or [Gly-Gly-Gly-Gly Ser]₃ (SEQ ID NO: 152).

In certain embodiments, charged linkers may be used. Such chargeslinkers may contain a significant number of acidic residues (e.g., Asp,Glu, and the like), or may contain a significant number of basisresidues (e.g., Lys, Arg, and the like), such that the linker has a pIlower than 7 or greater than 7, respectively. As understood by theartisan, and all other things being equal, the greater the relativeamount of acidic or basic residues in a given linker, the lower orhigher, respectively, the pI of the linker will be. Such linkers mayimpart advantages to the engineered polypeptides disclosed herein, suchas improving solubility and/or stability characteristics of suchpolypeptides at a particular pH, such as a physiological pH (e.g.,between pH 7.2 and pH 7.6, inclusive), or a pH of a pharmaceuticalcomposition comprising such polypeptides.

For example, an “acidic linker” is a linker that has a pI of less than7; between 6 and 7, inclusive; between 5 and 6, inclusive; between 4 and5, inclusive; between 3 and 4, inclusive; between 2 and 3, inclusive; orbetween 1 and 2, inclusive. Similarly, a “basic linker” is a linker thathas a pI of greater than 7; between 7 and 8, inclusive; between 8 and 9,inclusive; between 9 and 10, inclusive; between 10 and 11, inclusive;between 11 and 12 inclusive, or between 12 and 13, inclusive. In certainembodiments, an acidic linker will contain a sequence that is selectedfrom the group consisting of [Gly-Glu]_(n) (SEQ ID NO:126);[Gly-Gly-Glu]_(n) (SEQ ID NO:127); [Gly-Gly-Gly-Glu]_(n) (SEQ IDNO:128); [Gly-Gly-Gly-Gly-Glu]_(n) (SEQ ID NO:129), [Gly-Asp]_(n) (SEQID NO:130); [Gly-Gly-Asp]_(n) (SEQ ID NO:131); [Gly-Gly-Gly-Asp]_(n)(SEQ ID NO:132); [Gly-Gly-Gly-Gly-Asp]_(n) (SEQ ID NO:133) where n is 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more; for example, [Gly-Gly-Glu]₆ (SEQ IDNO: 153). In certain embodiments, a basic linker will contain a sequencethat is selected from the group consisting of [Gly-Lys]_(n) (SEQ IDNO:134); [Gly-Gly-Lys]_(n) (SEQ ID NO:135); [Gly-Gly-Gly-Lys]_(n) (SEQID NO:136); [Gly-Gly-Gly-Gly-Lys]_(n) (SEQ ID NO:137), [Gly-Arg]_(n)(SEQ ID NO:138); [Gly-Gly-Arg]_(n) (SEQ ID NO:139);[Gly-Gly-Gly-Arg]_(n) (SEQ ID NO:140); [Gly-Gly-Gly-Gly-Arg]_(n) (SEQ IDNO:141) where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more; for example,[Gly-Gly-Lys]₆ (SEQ ID NO: 154).

Additionally, linkers may be prepared which possess certain structuralmotifs or characteristics, such as an α helix. For example, such alinker may contain an sequence that is selected from the groupconsisting of [Glu-Ala-Ala-Ala-Lys]_(n) (SEQ ID NO:142), where n is 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more; for example, [Glu-Ala-Ala-Ala-Lys]₃(SEQ ID NO: 155), [Glu-Ala-Ala-Ala-Lys]₄ (SEQ ID NO: 156), or[Glu-Ala-Ala-Ala-Lys]₅ (SEQ ID NO: 157).

Additionally, a non-peptidic linker may be employed to serve as the L1moiety of an engineered polypeptide described herein. For example, asunderstood in the art, an exemplary non-peptide linker such as a PEGlinker may be so-employed. See, e.g., WO2000024782. In certainembodiments, such a PEG linker has a molecular weight of 100 Da to 1000kDa. In certain embodiments, such a PEG linker has a molecular weight of100 Da to 500 kDa. In certain embodiments, such a PEG linker has amolecular weight of 100 Da to 100 kDa. In certain embodiments, such aPEG linker has a molecular weight of 100 Da to 50 kDa. In certainembodiments, such a PEG linker has a molecular weight of 100 Da to 10kDa. In certain embodiments, such a PEG linker has a molecular weight of100 Da to 5 kDa. In certain embodiments, such a PEG linker has amolecular weight of 100 Da to 1 kDa. In certain embodiments, such a PEGlinker has a molecular weight of 100 Da to 500 Da.

It is also to be understood that linkers suitable for use in accordancewith the invention may possess one or more of the characteristics andmotifs described above. For example, a linker may comprise an acidiclinker as well as a structural motif, such as an alpha helix. Similarly,a linker may comprise a basic linker and a structural motif, such as analpha helix. A linker may comprise an acidic linker, a basic linker, anda structural motif, such as an α helix. Additionally, it is also to beunderstood that engineered polypeptides in accordance with the inventionmay possess more than one linker, and each such linker may possess oneor more of the characteristics described above.

The linkers described herein are exemplary, and linkers within the scopeof this invention may be much longer and may include other residues. Inone embodiment, expressly excluded are engineered polypeptides in whichthe leptin compound is linked directly to the ABD without a linker.

In some embodiments, the engineered polypeptide includes an ABD at theN-terminal, and a HD1 at the C-terminal. Conversely, in someembodiments, the engineered polypeptide includes an ABD at theC-terminal, and a HD1 at the N-terminal. In some embodiments, either theN-terminal or the C-terminal is a leptin, a leptin fragment, or a leptinanalog. Preferably, the ABD is at the N-terminus of a leptin compound.Further to embodiments which include an ABD and a HD1, the engineeredpolypeptide can have the structure ABD-HD1 or HD1-ABD (both read in theN-terminal to C-terminal orientation).

It is understood that absent an express indication of the N-terminusand/or C-terminus of a engineered polypeptide set forth herein, theengineered polypeptide is to be read in the N-terminus to C-terminusorientation. For example, where HD1 is a leptin or analog thereof, theterms HD1-ABD, HD1-ABD, HD1-ABD, and the like mean, in the absence of anexpress indication of the N-terminus and/or the C-terminus, that theleptin compound resides at the N-terminus of the engineered polypeptide,and the ABD resides at the C-terminus. Conversely, if the N-terminusand/or C-terminus is expressly indicated, then the engineeredpolypeptide is to be read according to the express indication of theterminii. For example, the terms HD1_(C-term)-ABD, HD1-L1-ABD_(N-term)and the like mean that the ABD resides at the N-terminus of theengineered polypeptide, and HD1 resides at the C-terminus.

In some embodiments of the above described engineered polypeptides, HD1is human leptin or metreleptin. In some further embodiments, HD1 is aleptin analog as described herein. In some embodiments, the leptinanalog is leptin A100, A300 or A500.

In some embodiments, the engineered polypeptide described herein has anaffinity for serum albumin which is different than the affinity of theABD polypeptide alone, i.e., in the absence of a conjugated hormonedomain. In order to obtain effective association, the engineeredpolypeptide can have a binding affinity for serum albumin such that thedissociation constant K_(D) is, for example, less than about 10⁻⁶ M,10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, 10⁻¹² M, 10⁻¹³ M, 10⁻¹⁴ M oreven 10⁻¹⁵ M. In some embodiments, the affinity is not excessively tightsuch that the engineered polypeptide can dissociate from the albumin andelicit a biological response, for example binding to a receptor, forexample, a leptin receptor. The affinity can be measured as described inPCT Published Appl. No. WO 2009/016043, preferably to human serumalbumin.

In some embodiments, an engineered polypeptide described herein issuperior to a corresponding compound having a different moiety that canextend plasma half-life (e.g., PEG or of Fc or albumin) conjugated witha hormone domain(s). In this context, the term “superior” refers to avariety of functional properties which could be weighed in theevaluation of a treatment for a disease or disorder. For example, theengineered polypeptide described herein could require less biologicallyactive (hormone domain) component, for example 1×, 2×, 3×, 4×, 5×, oreven less, than the corresponding compound having a different moietyconjugated with the hormone domain(s). For further example, theengineered polypeptide described herein could have higher potency, forexample, 1.5×, 2×, 3×, 4×, 5×, 10×, 20×, 50×, or even higher potency.

Engineered polypeptide compounds contemplated herein include thecompounds as set forth in Table 2 following.

TABLE 2 Selected engineered polypeptides Cmpd Sequence MW  1MLAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALK 21647.2LHILAALPTGGGGASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPGC (SEQ ID NO: 53)  2MLAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALK 22509.0LHILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDM LQQLDLSPGC (SEQ ID NO: 54)  3MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLE 21734.3FIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPGCTGGGGSASLAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 55)  4MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLE 22509.0FIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPGCTGGGGSGGGSGGGSGGGSASLAEAKVLANRELDKYGVSDFYKRLINKAKTVEGV EALKLHILAALP (SEQ ID NO: 56)  9MLAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALK 22984.4LHILAALPTGGGGSGGGSGGGSGGGSASISIEKIQADTKTLTKTIITRIIQLSTQNGVSTDQRVSGLDFIPGNQQFQNLADMDQTLAVYQQILSSLPMPDRTQISNDLENLRSLFALLATLKNCPFTRSDGLDTMEIWGGIVEESLYSTEVVTLDRLRKSLKNI EKQLDHIQGC (SEQ ID NO: 57) 12MLAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALK 22597.1LHILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQD MLWQLDLSPGC (SEQ ID NO: 58) 13MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLD 22597.1FIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGCTGGGGSGGGSGGGSGGGSASLAEAKVLANRELDKYGVSDFYKRLINKAKTVEG VEALKLHILAALP (SEQ ID NO: 59) 14MLAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALK 22592.9LHILAALPTGGGGSGGGSGGGSGGGSASPIQRVQDDTKTLIKTIITRINDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQDML RQLDRNPGC (SEQ ID NO: 60) 15MLAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALK 22576.7LHILAALPTGGGGSGGGSGGGSGGGSASPIQRVQDDTKTLIKTIITRINDISPPQGVSSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQDMLR QLDRNPGC (SEQ ID N0: 61) 16MLAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALK 21332.7LHILAALPTGPIQRVQDDTKTLIKTIITRINDISPPQGVCSPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQDMLRQLDRNPGC (SEQ ID NO: 62) 17MLAEAKVLANRELDKYGVSDYYKNIINRAKTVEGVRALK 22624.7LHILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQD MLWQLDLSPGC (SEQ ID NO: 63) 18MLAEAKVLANRELDKYGVSDYYKNIINRAKTVEGVRALK 22536.6LHILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDM LQQLDLSPGC (SEQ ID NO: 64) 19MLAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALK 22480.95LHILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDM LQQLDLSPGC (SEQ ID NO: 65) 20MLAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALK 24224.6LHILAALPTGLAEAAAKEAAAKEAAAKEAAAKEAAAKAAAASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPGC (SEQ ID NO: 66) 21MLAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALK 22876.9LHILAALPTGGEGGEGGEGGEGGEGGEASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQD MLQQLDLSPGC (SEQ ID NO: 67) 22MLAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALK 22871.2LHILAALPTGGKGGKGGKGGKGGKGGKASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSL QDMLQQLDLSPGC (SEQ ID NO: 68)23 MLAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALK 22583.6LHILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCSLPQASGLETLESLGEVLEASGYSTEVVALSRLQGSLQDIL QQLDLSPEC (SEQ ID NO: 69) 24MLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALT 21597.02LHILAALPTGGGGASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPGC (SEQ ID NO: 70) 25MLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALT 22458.80LHILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDM LQQLDLSPGC (SEQ ID NO: 71) 26MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLE 21684.10FIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPGCTGGGGSASLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALTLHILAALP (SEQ ID NO: 72) 27MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLE 22458.80FIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPGCTGGGGSGGGSGGGSGGGSASLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGV EALTLHILAALP (SEQ ID NO: 73) 28MLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALT 22934.18LHILAALPTGGGGSGGGSGGGSGGGSASISIEKIQADTKTLTKTIITRIIQLSTQNGVSTDQRVSGLDFIPGNQQFQNLADMDQTLAVYQQILSSLPMPDRTQISNDLENLRSLFALLATLKNCPFTRSDGLDTMEIWGGIVEESLYSTEVVTLDRLRKSLKNI EKQLDHIQGC (SEQ ID NO: 74) 29MLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALT 22546.92LHILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQD MLWQLDLSPGC (SEQ ID NO: 75) 30MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLD 22546.92FIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGCTGGGGSGGGSGGGSGGGSASLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEG VEALTLHILAALP (SEQ ID NO: 76) 31MLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALT 22544.03LHILAALPTGGGGSGGGSGGGSGGGSASPIQRVQDDTKTLIKTIITRINDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQDML RQLDRNPGC (SEQ ID NO: 77) 32MLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALT 22527.96LHILAALPTGGGGSGGGSGGGSGGGSASPIQRVQDDTKTLIKTIITRINDISPPQGVSSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQDMLR QLDRNPGC (SEQ ID NO: 78) 33MLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALT 21196.74LHILAALPTGPIQRVQDDTKTLIKTIITRINDISPPQGVCSPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQDMLRQLDRNPGC (SEQ ID NO: 79) 34MLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALT 22546.92LHILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQD MLWQLDLSPGC (SEQ ID NO: 80) 35MLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALT 22458.80LHILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDM LQQLDLSPGC (SEQ ID NO: 81) 36MLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALT 22430.75LHILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDM LQQLDLSPGC (SEQ ID NO: 82) 37MLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALT 24175.99LHILAALPTGLAEAAAKEAAAKEAAAKEAAAKEAAAKAAAASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPGC (SEQ ID NO: 83) 38MLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALT 22828.13LHILAALPTGGEGGEGGEGGEGGEGGEASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQD MLQQLDLSPGC (SEQ ID NO: 84) 39MLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALT 22836.51LHILAALPTGGKGGKGGKGGKGGKGGKASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSL QDMLQQLDLSPGC (SEQ ID NO: 85)40 MLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALT 22534.83LHILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCSLPQASGLETLESLGEVLEASGYSTEVVALSRLQGSLQDIL QQLDLSPEC (SEQ ID NO: 86) 41MLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALIS 21574.96EILAALPTGGGGASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPGC (SEQ ID NO: 87) 42MLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALIS 22436.75EILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDML QQLDLSPGC (SEQ ID NO: 88) 43MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLE 21662.04FIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPGCTGGGGSASLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALISEILAALP (SEQ ID NO: 89) 44MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLE 22436.75FIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPGCTGGGGSGGGSGGGSGGGSASLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGV EALISEILAALP (SEQ ID NO: 90) 45MLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALIS 22912.13EILAALPTGGGGSGGGSGGGSGGGSASISIEKIQADTKTLTKTIITRIIQLSTQNGVSTDQRVSGLDFIPGNQQFQNLADMDQTLAVYQQILSSLPMPDRTQISNDLENLRSLFALLATLKNCPFTRSDGLDTMEIWGGIVEESLYSTEVVTLDRLRKSLKNIE KQLDHIQGC (SEQ ID NO: 91) 46MLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALIS 22524.86EILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQD MLWQLDLSPGC (SEQ ID NO: 92) 47MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLD 22524.86FIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGCTGGGGSGGGSGGGSGGGSASLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEG VEALISEILAALP (SEQ ID NO: 93) 48MLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALIS 22521.97EILAALPTGGGGSGGGSGGGSGGGSASPIQRVQDDTKTLIKTIITRINDISPPQGVCSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQDMLR QLDRNPGC (SEQ ID NO: 94) 49MLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALIS 22505.91EILAALPTGGGGSGGGSGGGSGGGSASPIQRVQDDTKTLIKTIITRINDISPPQGVSSRPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQDMLR QLDRNPGC (SEQ ID NO: 95) 50MLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALIS 21174.68EILAALPTGPIQRVQDDTKTLIKTIITRINDISPPQGVCSPRVAGLDFIPRVQSVRTLSGMDQILATYQQILTSLQSRNVIQISNDLENLRDLLHVLAFSKSCPVPRARGSDTIKGLGNVLRASVHSTEVVALSRLKAALQDMLRQLDRNPGC (SEQ ID NO: 96) 51MLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALIS 22524.86EILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQD MLWQLDLSPGC (SEQ ID NO: 97) 52MLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALIS 22436.75EILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDML QQLDLSPGC (SEQ ID NO: 98) 53MLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALIS 22408.70EILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDM LQQLDLSPGC (SEQ ID NO: 99) 54MLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALIS 24153.93EILAALPTGLAEAAAKEAAAKEAAAKEAAAKEAAAKAAAASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDMLQQLDLSPGC (SEQ ID NO: 100) 55MLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALIS 22806.08EILAALPTGGEGGEGGEGGEGGEGGEASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDM LQQLDLSPGC (SEQ ID NO: 101) 56MLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALIS 22800.43EILAALPTGGKGGKGGKGGKGGKGGKASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQD MLQQLDLSPGC (SEQ ID NO: 102) 58MLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALIS 22512.78EILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCSLPQASGLETLESLGEVLEASGYSTEVVALSRLQGSLQDILQ QLDLSPEC (SEQ ID NO: 103) 59MLAEAKVLANRELDKYGVSDYYKNLINKAKTVEGVEAL 22455.85TLHILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQD MLQQLDLSPGC (SEQ ID NO: 104) 60MLAEAKEDAIKELDKYGVSDYYKRLISKAKTVEGVKALIS 22450.87EILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDML QQLDLSPGC (SEQ ID NO: 105) 61MLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALT 22458.80LHILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDM LQQLDLSPGC (SEQ ID NO: 106) 62MLAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALIS 22436.75EILAALPTGGGGSGGGSGGGSGGGSASVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLEFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPQASGLETLESLGGVLEASGYSTEVVALSRLQGSLQDML QQLDLSPGC (SEQ ID NO: 107)

Specifically contemplated are compounds of the above sequences in whichthe N-terminal methionine is absent, e.g. where the N-terminal commenceswith VPIQKV (SEQ ID NO: 158) or LAEAK (SEQ ID NO: 159) for example, forleptin compounds The N-terminal methionine is present primarily as aconvenience for bacterial expression. However, conjugate peptides of thepresent invention can be expressed in a eukaryotic host cell (e.g. yeast(e.g. Pichia), mammalian, baculovirus) or other host cell havingpost-translational N-terminal proteolytic processing to yield anN-terminal amino acid as found in a naturally occurring mature peptidecounterpart of the desired hormone or ABD sequence. Alternatively, anN-terminal sequence used for expression and/or secretion can be one thatcan be removed post-translationally, e.g. as by use of a protease suchas TEV.

III. Methods of Design and Production

Design of Constructs.

The engineered polypeptides described herein can be designed at theamino acid level. These sequences can then be back translated using avariety of software products known in the art such that the nucleotidesequence is optimized for the desired expression host, e.g. basedprotein expression, codon optimization, restriction site content. Forexample, the nucleotide sequence can be optimized for E. coli basedprotein expression and for restriction site content. Based on thenucleotide sequence of interest, overlapping oligonucleotides can beprovided for multistep PCR, as known in the art. These oligonucleotidescan be used in multiple PCR reactions under conditions well known in theart to build the cDNA encoding the protein of interest. For one exampleis 1× Amplitaq Buffer, 1.3 mM MgCl₂, 200 uM dNTPs, 4 U Amplitaq Gold,0.2 uM of each primer (AmpliTaq Gold, ABI), with cycling parameters: (94C:30 s, 58 C:1 min, 72 C:1 min), 35 cycles.

Restriction sites can be added to the ends of the PCR products for usein vector ligation as known in the art. Specific sites can include Nde1and Xho1, such that the cDNA can then be in the proper reading frame ina pET45b expression vector (Novagen). By using these sites, anyN-terminal His Tag that are in this vector can be removed as thetranslation start site would then be downstream of the tag. Onceexpression constructs are completed, verification can be conduct bysequencing using e.g., T7 promoter primer, T7 terminator primer andstandard ABI BigDye Term v3.1 protocols as known in the art. Sequenceinformation can be obtained from e.g., an ABI 3730 DNA Analyzer and canbe analyzed using Vector NTI v.10 software (Invitrogen). Expressionconstructs can be designed in a modular manner such that linkersequences can be easily cut out and changed, as known in the art.

Protease recognition sites, known in the art or described herein, can beincorporated into constructs useful for the design, construction,manipulation and production of recombinant engineering polypeptidesdescribed herein.

General Methods of Production.

The engineered polypeptides described herein may be prepared usingbiological, chemical, and/or recombinant DNA techniques that are knownin the art. Exemplary methods are described herein and in U.S. Pat. No.6,872,700; WO 2007/139941; WO 2007/140284; WO 2008/082274; WO2009/011544; and US Publication No. 2007/0238669, the disclosures ofwhich are incorporated herein by reference in their entireties and forall purposes. Other methods for preparing the compounds are set forthherein.

The engineered polypeptides described herein may be prepared usingstandard solid-phase peptide synthesis techniques, such as an automatedor semiautomated peptide synthesizer. Typically, using such techniques,an alpha-N-carbamoyl protected amino acid and an amino acid attached tothe growing peptide chain on a resin are coupled at RT in an inertsolvent (e.g., dimethylformamide, N-methylpyrrolidinone, methylenechloride, and the like) in the presence of coupling agents (e.g.,dicyclohexylcarbodiimide, 1-hydroxybenzo-triazole, and the like) in thepresence of a base (e.g., diisopropylethylamine, and the like). Thealpha-N-carbamoyl protecting group is removed from the resultingpeptide-resin using a reagent (e.g., trifluoroacetic acid, piperidine,and the like) and the coupling reaction repeated with the next desiredN-protected amino acid to be added to the peptide chain. SuitableN-protecting groups are well known in the art, such ast-butyloxycarbonyl (tBoc) fluorenylmethoxycarbonyl (Fmoc), and the like.The solvents, amino acid derivatives and 4-methylbenzhydryl-amine resinused in the peptide synthesizer may be purchased from Applied BiosystemsInc. (Foster City, Calif.).

For chemical synthesis solid phase peptide synthesis can be used for theengineered polypeptides, since in general solid phase synthesis is astraightforward approach with excellent scalability to commercial scale,and is generally compatible with relatively long engineeredpolypeptides. Solid phase peptide synthesis may be carried out with anautomatic peptide synthesizer (Model 430A, Applied Biosystems Inc.,Foster City, Calif.) using the NMP/HOBt (Option 1) system and tBoc orFmoc chemistry (See Applied Biosystems User's Manual for the ABI 430APeptide Synthesizer, Version 1.3B Jul. 1, 1988, section 6, pp. 49-70,Applied Biosystems, Inc., Foster City, Calif.) with capping.Boc-peptide-resins may be cleaved with HF (−5° C. to 0° C., 1 hour). Thepeptide may be extracted from the resin with alternating water andacetic acid, and the filtrates lyophilized. The Fmoc-peptide resins maybe cleaved according to standard methods (e.g., Introduction to CleavageTechniques, Applied Biosystems, Inc., 1990, pp. 6-12). Peptides may alsobe assembled using an Advanced Chem Tech Synthesizer (Model MPS 350,Louisville, Ky.).

The compounds described herein may also be prepared using recombinantDNA techniques using methods known in the art, such as Sambrook et al.,1989, MOLECULAR CLONING: A LABORATORY MANUAL, 2d Ed., Cold SpringHarbor. Non-peptide compounds may be prepared by art-known methods. Forexample, phosphate-containing amino acids and peptides containing suchamino acids, may be prepared using methods known in the art, such asdescribed in Bartlett et al, 1986, Biorg. Chem. 14:356-377.

The engineered polypeptides may alternatively be produced by recombinanttechniques well known in the art. See, e.g., Sambrook et al., 1989(Id.). These engineered polypeptides produced by recombinanttechnologies may be expressed from a polynucleotide. One skilled in theart will appreciate that the polynucleotides, including DNA and RNA,that encode such engineered polypeptides may be obtained from thewild-type cDNA, e.g. human leptin, taking into consideration thedegeneracy of codon usage, and may further engineered as desired toincorporate the indicated substitutions. These polynucleotide sequencesmay incorporate codons facilitating transcription and translation ofmRNA in microbial hosts. Such manufacturing sequences may readily beconstructed according to the methods well known in the art. See, e.g.,WO 83/04053, incorporated herein by reference in its entirety and forall purposes. The polynucleotides above may also optionally encode anN-terminal methionyl residue. Non-peptide compounds useful in thepresent invention may be prepared by art-known methods. For example,phosphate-containing amino acids and peptides containing such aminoacids may be prepared using methods known in the art. See, e.g.,Bartlett and Landen, 1986, Bioorg. Chem. 14: 356-77.

A variety of expression vector/host systems may be utilized to containand express a engineered polypeptide coding sequence. These include butare not limited to microorganisms such as bacteria transformed withrecombinant bacteriophage, plasmid or cosmid DNA expression vectors;yeast transformed with yeast expression vectors; insect cell systemsinfected with virus expression vectors (e.g., baculovirus); plant cellsystems transfected with virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withbacterial expression vectors (e.g., T1 or pBR322 plasmid); or animalcell systems. Mammalian cells that are useful in recombinant proteinproductions include but are not limited to VERO cells, HeLa cells,Chinese hamster ovary (CHO) cell lines, COS cells (such as COS-7), WI38, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and 293 cells.Exemplary protocols for the recombinant expression of the protein aredescribed herein and/or are known in the art.

As such, polynucleotide sequences are useful in generating new anduseful viral and plasmid DNA vectors, new and useful transformed andtransfected prokaryotic and eukaryotic host cells (including bacterial,yeast, and mammalian cells grown in culture), and new and useful methodsfor cultured growth of such host cells capable of expression of thepresent engineered polypeptides. The polynucleotide sequences encodingengineered polypeptides herein may be useful for gene therapy ininstances where underproduction of engineered polypeptides would bealleviated, or the need for increased levels of such would be met.

The present invention also provides for processes for recombinant DNAproduction of the present engineered polypeptides. Provided is a processfor producing the engineered polypeptides from a host cell containingnucleic acids encoding the engineered polypeptide comprising: (a)culturing the host cell containing polynucleotides encoding theengineered polypeptide under conditions facilitating the expression ofthe DNA molecule; and (b) obtaining the engineered polypeptide.

Host cells may be prokaryotic or eukaryotic and include bacteria,mammalian cells (such as Chinese Hamster Ovary (CHO) cells, monkeycells, baby hamster kidney cells, cancer cells or other cells), yeastcells, and insect cells.

Mammalian host systems for the expression of the recombinant proteinalso are well known to those of skill in the art. Host cell strains maybe chosen for a particular ability to process the expressed protein orproduce certain post-translation modifications that will be useful inproviding protein activity. Such modifications of the polypeptideinclude, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation.Post-translational processing, which cleaves a “prepro” form of theprotein, may also be important for correct insertion, folding and/orfunction. Different host cells, such as CHO, HeLa, MDCK, 293, W138, andthe like, have specific cellular machinery and characteristic mechanismsfor such post-translational activities, and may be chosen to ensure thecorrect modification and processing of the introduced foreign protein.

Alternatively, a yeast system may be employed to generate the engineeredpolypeptides of the present invention. The coding region of theengineered polypeptides DNA is amplified by PCR. A DNA encoding theyeast pre-pro-alpha leader sequence is amplified from yeast genomic DNAin a PCR reaction using one primer containing nucleotides 1-20 of thealpha mating factor gene and another primer complementary to nucleotides255-235 of this gene (Kurjan and Herskowitz, 1982, Cell, 30:933-43). Thepre-pro-alpha leader coding sequence and engineered polypeptide codingsequence fragments are ligated into a plasmid containing the yeastalcohol dehydrogenase (ADH2) promoter, such that the promoter directsexpression of a fusion protein consisting of the pre-pro-alpha factorfused to the mature engineered polypeptide. As taught by Rose andBroach, Meth. Enz. 185: 234-79, Goeddel ed., Academic Press, Inc., SanDiego, Calif. (1990), the vector further includes an ADH2 transcriptionterminator downstream of the cloning site, the yeast “2-micron”replication origin, the yeast leu-2d gene, the yeast REP1 and REP2genes, the E. coli beta-lactamase gene, and an E. coli origin ofreplication. The beta-lactamase and leu-2d genes provide for selectionin bacteria and yeast, respectively. The leu-2d gene also facilitatesincreased copy number of the plasmid in yeast to induce higher levels ofexpression. The REP1 and REP2 genes encode proteins involved inregulation of the plasmid copy number.

The DNA construct described in the preceding paragraph is transformedinto yeast cells using a known method, e.g., lithium acetate treatment(Steams et al., 1990,. Meth. Enz. 185: 280-297). The ADH2 promoter isinduced upon exhaustion of glucose in the growth media (Price et al.,1987, Gene 55:287). The pre-pro-alpha sequence effects secretion of thefusion protein from the cells. Concomitantly, the yeast KEX2 proteincleaves the pre-pro sequence from the mature engineered polypeptides(Bitter et al., 1984, Proc. Natl. Acad. Sci. USA 81:5330-5334).

Engineered polypeptides of the invention may also be recombinantlyexpressed in yeast, e.g., Pichia, using a commercially availableexpression system, e.g., the Pichia Expression System (Invitrogen, SanDiego, Calif.), following the manufacturer's instructions. This systemalso relies on the pre-pro-alpha sequence to direct secretion, buttranscription of the insert is driven by the alcohol oxidase (AOX1)promoter upon induction by methanol. The secreted engineered polypeptideis purified from the yeast growth medium by, e.g., the methods used topurify said engineered polypeptide from bacterial and mammalian cellsupernatants.

Alternatively, the DNA encoding a engineered polypeptide may be clonedinto a baculovirus expression vector, e.g. pVL1393 (PharMingen, SanDiego, Calif.). This engineered-polypeptide-encoding vector is then usedaccording to the manufacturer's directions (PharMingen) or knowntechniques to infect Spodoptera frugiperda cells, grown for example insF9 protein-free media, and to produce recombinant protein. The proteinis purified and concentrated from the media using methods known in theart, e.g. a heparin-Sepharose column (Pharmacia, Piscataway, N.J.) andsequential molecular sizing columns (Amicon, Beverly, Mass.), andresuspended in appropriate solution, e.g. PBS. SDS-PAGE analysis can beused to characterize the protein, for example by showing a single bandthat confirms the size of the desired engineered polypeptide, as canfull amino acid amino acid sequence analysis, e.g. Edman sequencing on aProton 2090 Peptide Sequencer, or confirmation of its N-terminalsequence.

For example, the DNA sequence encoding the predicted mature engineeredpolypeptide may be cloned into a plasmid containing a desired promoterand, optionally, a leader sequence (see, e.g., Better et al., 1988,Science 240:1041-1043). The sequence of this construct may be confirmedby automated sequencing. The plasmid is then transformed into E. coli,strain MC1061, using standard procedures employing CaCl₂ incubation andheat shock treatment of the bacteria (Sambrook et al., Id.). Thetransformed bacteria are grown in LB medium supplemented withcarbenicillin, and production of the expressed protein is induced bygrowth in a suitable medium. If present, the leader sequence will affectsecretion of the mature engineered polypeptide and be cleaved duringsecretion. The secreted recombinant engineered polypeptide is purifiedfrom the bacterial culture media by the method described herein.

Alternatively, the engineered polypeptides may be expressed in an insectsystem. Insect systems for protein expression are well known to those ofskill in the art. In one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia larvae. The engineeredpolypeptide coding sequence is cloned into a nonessential region of thevirus, such as the polyhedrin gene, and placed under control of thepolyhedrin promoter. Successful insertion of a engineered polypeptidewill render the polyhedrin gene inactive and produce recombinant viruslacking coat protein coat. The recombinant viruses are then used toinfect S. frugiperda cells or Trichoplusia larvae in which engineeredpolypeptide of the present invention is expressed (Smith et al., 1983,J. Virol. 46:584; Engelhard et al., 1994, Proc. Natl. Acad. Sci. USA91:3224-3227).

In another example, the DNA sequence encoding the engineeredpolypeptides may be amplified by PCR and cloned into an appropriatevector, for example, pGEX-3X (Pharmacia, Piscataway, N.J.). The pGEXvector is designed to produce a fusion protein comprisingglutathione-S-transferase (GST), encoded by the vector, and a proteinencoded by a DNA fragment inserted into the vector's cloning site. Theprimers for the PCR may be generated to include, for example, anappropriate cleavage site. The recombinant fusion protein may then becleaved from the GST portion of the fusion protein. ThepGEX-3X/engineered polypeptide construct is transformed into E. coliXL-1 Blue cells (Stratagene, La Jolla, Calif.), and individualtransformants are isolated and grown at 37° C. in LB medium(supplemented with carbenicillin) to an optical density at wavelength600 nm of 0.4, followed by further incubation for 4 hours in thepresence of 0.5 mM Isopropyl beta-D-thiogalactopyranoside (SigmaChemical Co., St. Louis, Mo.). Plasmid DNA from individual transformantsis purified and partially sequenced using an automated sequencer toconfirm the presence of the desired engineered polypeptide-encoding geneinsert in the proper orientation.

The fusion protein, when expected to be produced as an insolubleinclusion body in the bacteria, may be purified as described above or asfollows. Cells are harvested by centrifugation; washed in 0.15 M NaCl,10 mM Tris, pH 8, 1 mM EDTA; and treated with 0.1 mg/mL lysozyme (SigmaChemical Co.) for 15 min. at RT. The lysate is cleared by sonication,and cell debris is pelleted by centrifugation for 10 min. at 12,000×g.The fusion protein-containing pellet is resuspended in 50 mM Tris, pH 8,and 10 mM EDTA, layered over 50% glycerol, and centrifuged for 30 min.at 6000×g. The pellet is resuspended in standard phosphate bufferedsaline solution (PBS) free of Mg++ and Ca++. The fusion protein isfurther purified by fractionating the resuspended pellet in a denaturingSDS polyacrylamide gel (Sambrook et al., supra). The gel is soaked in0.4 M KCl to visualize the protein, which is excised and electroelutedin gel-running buffer lacking SDS. If the GST/engineered polypeptidefusion protein is produced in bacteria as a soluble protein, it may bepurified using the GST Purification Module (Pharmacia Biotech).

The fusion protein may be subjected to digestion to cleave the GST fromthe mature engineered polypeptide. The digestion reaction (20-40 μgfusion protein, 20-30 units human thrombin (4000 U/mg (Sigma) in 0.5 mLPBS) is incubated 16-48 hrs. at RT and loaded on a denaturing SDS-PAGEgel to fractionate the reaction products. The gel is soaked in 0.4 M KClto visualize the protein bands. The identity of the protein bandcorresponding to the expected molecular weight of the engineeredpolypeptide may be confirmed by partial amino acid sequence analysisusing an automated sequencer (Applied Biosystems Model 473A, FosterCity, Calif.).

In a particularly exemplary method of recombinant expression of theengineered polypeptides of the present invention, 293 cells may beco-transfected with plasmids containing the engineered polypeptides cDNAin the pCMV vector (5′ CMV promoter, 3′ HGH poly A sequence) and pSV2neo(containing the neo resistance gene) by the calcium phosphate method.

In one embodiment, the vectors should be linearized with ScaI prior totransfection. Similarly, an alternative construct using a similar pCMVvector with the neo gene incorporated can be used. Stable cell lines areselected from single cell clones by limiting dilution in growth mediacontaining 0.5 mg/mL G418 (neomycin-like antibiotic) for 10-14 days.Cell lines are screened for engineered polypeptides expression by ELISAor Western blot, and high-expressing cell lines are expanded for largescale growth.

It is preferable that the transformed cells are used for long-term,high-yield protein production and as such stable expression isdesirable. Once such cells are transformed with vectors that containselectable markers along with the desired expression cassette, the cellsmay be allowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The selectable marker is designed to conferresistance to selection, and its presence allows growth and recovery ofcells that successfully express the introduced sequences. Resistantclumps of stably transformed cells can be proliferated using tissueculture techniques appropriate to the cell.

A number of selection systems may be used to recover the cells that havebeen transformed for recombinant protein production. Such selectionsystems include, but are not limited to, HSV thymidine kinase,hypoxanthine-guanine phosphoribosyltransferase and adeninephosphoribosyltransferase genes, in tk-, hgprt- or aprt-cells,respectively. Also, anti-metabolite resistance can be used as the basisof selection for dhfr, that confers resistance to methotrexate; gpt,that confers resistance to mycophenolic acid; neo, that confersresistance to the aminoglycoside, G418; also, that confers resistance tochlorsulfuron; and hygro, that confers resistance to hygromycin.Additional selectable genes that may be useful include trpB, whichallows cells to utilize indole in place of tryptophan, or hisD, whichallows cells to utilize histinol in place of histidine. Markers thatgive a visual indication for identification of transformants includeanthocyanins, beta-glucuronidase and its substrate, GUS, and luciferaseand its substrate, luciferin.

The engineered polypeptides of the present invention may be producedusing a combination of both automated peptide synthesis and recombinanttechniques. For example, either or both of: the leptin; a leptin analog,a active leptin fragment, or leptin derivative; and an ABD; andoptionally a linker; employed in the preparation of the engineeredpolypeptides as disclosed herein can be made synthetically orrecombinantly and then ligated together using methods known in the art,such as “native chemical ligation” and known variations thereof in whichan amide bond is formed joining the parent compounds. See for exampleU.S. Pat. No. 6,326,468, which is incorporated herein by reference foral purposes. Alternatively, for example, an engineered polypeptide ofthe present invention may contain a combination of modificationsincluding deletion, substitution, insertion and derivatization byPEGylation (or other moiety, e.g. polymer, fatty acyl chain, C-terminalamidation). Such a engineered polypeptide may be produced in stages. Inthe first stage, an intermediate engineered polypeptide containing themodifications of deletion, substitution, insertion, and any combinationthereof, may be produced by recombinant techniques as described. Thenafter an optional purification step as described herein, theintermediate engineered polypeptide is PEGylated (or subjected to otherchemical derivatization, e.g., acylation, C-terminal amidation) throughchemical modification with an appropriate PEGylating reagent (e.g., fromNeKtar Transforming Therapeutics, San Carlos, Calif.) to yield thedesired engineered polypeptide derivative. One skilled in the art willappreciate that the above-described procedure may be generalized toapply to a engineered polypeptide containing a combination ofmodifications selected from deletion, substitution, insertion,derivation, and other means of modification well known in the art andcontemplated by the present invention.

C-terminal amidation can be achieved by use of a glycine aminoacid-C-terminally extended precursor, synthesized for example in yeast(e.g. Pichia) as alpha-factor fusion protein that will be secreted intoculture medium. After purification, the C-terminal glycine of theengineered polypeptide precursor can be converted to amide by enzymaticamidation, e.g. peptidylglycine alpha-amidating monooxygenase (PAM). Seee.g., Cooper et al., 1989, Biochem. Biophys. Acta, 1014:247-258. Seealso U.S. Pat. No. 6,319,685, which is incorporated herein by referencein its entirety and for all purposes, which teaches methods forenzymatic amidation, including an alpha-amidating enzyme from rat beingsufficiently pure in alpha-amidating enzyme to exhibit a specificactivity of at least about 25 mU per mg of protein, and beingsufficiently free of proteolytic impurities to be suitable for use withsubstrates purified from natural sources or produced by recombinant DNAtechniques.

Peptides may be purified by any number of methods known in the art,including as described herein In one method peptides are purified byRP-HPLC (preparative and analytical) using a Waters Delta Prep 3000system. A C4, C8 or C18 preparative column (10μ, 2.2×25 cm; Vydac,Hesperia, Calif.) may be used to isolate peptides, and purity may bedetermined using a C4, C8 or C18 analytical column (5μ, 0.46×25 cm;Vydac). Solvents (A=0.1% TFA/water and B=0.1% TFA/CH₃CN) may bedelivered to the analytical column at a flow rate of 1.0 ml/min and tothe preparative column at 15 ml/min. Amino acid analyses may beperformed on the Waters Pico Tag system and processed using the Maximaprogram. Peptides may be hydrolyzed by vapor-phase acid hydrolysis (115°C., 20-24 h). Hydrolysates may be derivatized and analyzed by standardmethods (Cohen et al, THE PICO TAG METHOD: A MANUAL OF ADVANCEDTECHNIQUES FOR AMINO ACID ANALYSIS, pp. 11-52, Millipore Corporation,Milford, Mass. (1989)). Fast atom bombardment analysis may be carriedout by M-Scan, Incorporated (West Chester, Pa.). Mass calibration may beperformed using cesium iodide or cesium iodide/glycerol. Plasmadesorption ionization analysis using time of flight detection may becarried out on an Applied Biosystems Bio-Ion 20 mass spectrometer.

Engineered Polypeptide Expression Assay.

Methods are available for assaying the level of protein expression by ahost cell. Procedures useful for assaying the level of proteinexpression by a host cell are exemplified in the following typicalprotocol. About 25 μl BL21 E. coli cells are transformed with 2 ulplasmid DNA (expression vector for the engineered polynucleotide). Cellscan be plated and incubated overnight at 37 degrees C. or at roomtemperature (RT) over a 48-hr period. A single colony can be selectedand used to grow starter culture in 4 ml LB media with appropriateantibiotic for ˜6 hrs. Glycerol stocks can be prepared by adding 100 ul80% sterile glycerol to 900 ul stock, which can then be mixed gently andstored at −80 C. A 250 μl sample can be removed for TCP uninducedsample. An aliquot, for example, 2 ml of Magic media containingappropriate antibiotic can be inoculated with 5 μl starter culture,which can then be incubated overnight (up to 24 hrs) at 37 C, 300 rpm.As known in the art, Magic Media is autoinducing. Alternatively, 60 mlMagic Media containing appropriate antibiotic can be inoculated with 60μl starter culture in a 250 ml or 125 ml Thompson flask, which can thenbe incubated overnight (up to 24 hrs) at 30 C, 300 rpm. Afterincubation, 250 μl culture can be removed from each tube and the cellspelleted. The cell can be resuspended in 1 ml 50 mM Tris pH 8, 150 mMNaCl, to which can be added 0.1 volumes (100 ul) POP culture reagent and1 μl r-lysozyme (1:750 dilution in r-lysozyme buffer). The mixture canbe mixed well and incubated at least 10 min at RT. The preparation canthen be centrifuge 10 min at 14000×G. The supernatant (soluble fraction)can be removed and retained, and samples can be prepared for gelanalysis (15 μl+5 μl LDS). The remaining inclusion body pellet can beresuspended in 1 ml 1% SDS with sonication. The sample can be preparedfor gel analysis (15 ul+5 μl LDS). For uninduced samples, 1.0 volumesPOP culture reagent and 1 μl r-lysozyme (1:750 dilution in r-lysozymebuffer) can be added. The mixture can be mixed well and incubated atleast 10 min at RT. These samples may not need to be centrifuged. Thesample can then be prepared for gel analysis (15 μl+5 μl LDS). NU-PAGEgels (4-12%) non-reduced in 1×MES buffer can be run and stained withSimplyBlue microwave protocol. Destaining can be conducted overnight, asknown in the art. A gel image can be retained, and analyzed to determineprotein expression levels.

Inclusion Body Preparation.

For engineered polypeptides that are found in the inclusion bodyfraction, the following procedure can be beneficial. The cell pellet canbe resuspended in a minimum of 100 ml Lysis buffer for each 50 mlculture. Upon the addition of 30 ml, a 10 ml pipette can be used toresuspend, then the tube can be washed out with an additional 70 ml. Theresuspended cell solution can be multiply run, e.g., 4 passes, through amicrofluidizer at 100 PSI (min) taking care to keep chamber in ice waterthrough the entire process. The fluidized slurry can be centrifuged at14000×g, 20 min (e.g., JLA 10.5, 10,000 rpm, using 250 ml Nalgene®bottles). The inclusion body pellet can be resuspended on ice in chilledlysis buffer with stir bar and stir plate for 1 hour at 4 C afterdisruption with pipette tip. The pellet can be resuspended a second timein distilled H₂O with stir bar and stir plate for 1 hour at 4 C afterdisruption with pipette tip, followed by centrifugation at 14000×g, 15min. The supernatant can be removed and discarded. The resultant can bestored at −80 C.

Protein Purification.

As described herein, numerous methods are known for isolation ofexpressed polypeptides. The following is one example. Inclusion bodypellets can be solubilized in appropriate volume of solubilizationbuffer (8M urea or 8M guanidine, 50 mM Tris, 10 mM DTT, pH 7.75) for 1hour at RT. The solubilized pellets can be centrifuged for 20 min at 27000 g. Filtered (e.g., 0.4 um) supernatant can be transferred drop bydrop into appropriate volume of refolding buffer (50 mM Tris-HCl, 1 Murea, 0.8 M arginine, 4 mM cysteine, 1 mM cystamine; pH 8) at RT. Theresult can then be placed at 4° C. overnight or longer with gentlemixing. Samples can be concentrated and run on a gel filtration column(Superdex™ 75 26/60) at 1-2 ml/min in 4 C environment using a GEHealthsciences AKTAFPLC™. Appropriate protein containing fractions canbe identified via SDS-PAGE, pooled and run through a second gelfiltration column. Pooled protein can then be concentrated in Amiconfilter to appropriate concentration and assayed for endotoxin levelsusing, e.g., Endosafe® PTS Reader (Charles River), as known in the art.Once a protein sample has passed the endotoxin criteria, it can besterile filtered, dispensed into aliquots and run through qualitycontrol assays. Quality control assays can include analytical HPLC-SEC,non reducing SDS PAGE and RP HPLC-MS to obtain approximate mass.Proteins can be obtained in 1×PBS (137 mM sodium chloride, 2.7 mMpotassium chloride, 4.3 mM disodium phosphate, 1.4 mM monopotassiumphosphate, pH7.2), distributed into aliquots and flash frozen forstorage at −70 to −80° C.

IV. Methods of Use and Treating Disease

Indications.

A variety of diseases and disorders are contemplated to be beneficiallytreated by the polypeptide compounds and methods described herein.

Obesity and Overweight.

Obesity and its associated disorders including overweight are common andserious public health problems in the United States and throughout theworld. Upper body obesity is the strongest risk factor known for type 2diabetes mellitus and is a strong risk factor for cardiovasculardisease. Obesity is a recognized risk factor for hypertension,atherosclerosis, congestive heart failure, stroke, gallbladder disease,osteoarthritis, sleep apnea, reproductive disorders such as polycysticovarian syndrome, cancers of the breast, prostate, and colon, andincreased incidence of complications of general anesthesia. See, e.g.,Kopelman, 2000, Nature 404:635-43.

Obesity reduces life-span and carries a serious risk of theco-morbidities listed above, as well disorders such as infections,varicose veins, acanthosis nigricans, eczema, exercise intolerance,insulin resistance, hypertension hypercholesterolemia, cholelithiasis,orthopedic injury, and thromboembolic disease. See e.g., Rissanen et al,1990, Br. Med. J., 301:835-7. Obesity is also a risk factor for thegroup of conditions called insulin resistance syndrome, or “Syndrome X”and metabolic syndrome. The worldwide medical cost of obesity andassociated disorders is enormous.

The pathogenesis of obesity is believed to be multi-factoral. A problemis that, in obese subjects, nutrient availability and energy expendituredo not come into balance until there is excess adipose tissue. Thecentral nervous system (CNS) controls energy balance and coordinates avariety of behavioral, autonomic and endocrine activities appropriate tothe metabolic status of the animal. The mechanisms or systems thatcontrol these activities are broadly distributed across the forebrain(e.g., hypothalamus), hindbrain (e.g., brainstem), and spinal cord.Ultimately, metabolic (i.e., fuel availability) and cognitive (i.e.,learned preferences) information from these systems is integrated andthe decision to engage in appetitive (food seeking) and consummatory(ingestion) behaviors is either turned on (meal procurement andinitiation) or turned off (meal termination). The hypothalamus isthought to be principally responsible for integrating these signals andthen issuing commands to the brainstem. Brainstem nuclei that controlthe elements of the consummatory motor control system (e.g., musclesresponsible for chewing and swallowing). As such, these CNS nuclei haveliterally been referred to as constituting the “final common pathway”for ingestive behavior.

Neuroanatomical and pharmacological evidence support that signals ofenergy and nutritional homeostasis integrate in forebrain nuclei andthat the consummatory motor control system resides in brainstem nuclei,probably in regions surrounding the trigeminal motor nucleus. There areextensive reciprocal connection between the hypothalamus and brainstem.A variety of CNS-directed anti-obesity therapeutics (e.g., smallmolecules and peptides) focus predominantly upon forebrain substratesresiding in the hypothalamus and/or upon hindbrain substrates residingin the brainstem.

Obesity remains a poorly treatable, chronic, essentially intractablemetabolic disorder. Accordingly, a need exists for new therapies usefulin weight reduction and/or weight maintenance in a subject. Suchtherapies would lead to a profound beneficial effect on the subject'shealth. Methods and therapies employing the engineered peptidesdisclosed herein, either alone or in combination with other anti-obesityagents (see, e.g., WO 2009064298 and US 20080207512 may provide suchbeneficial effects.

Leptin Deficiency.

Leptin deficiency has been shown to result in obesity. One form ofleptin deficiency is congenital leptin deficiency, a rare geneticdisorder. See Montague et al., 1997, Nature 387: 903-908. Severe leptindeficiency can be a result of uncontrolled insulin-deficient diabetesmellitus that results from destruction of insulin-secreting β-cells. Itis theorized that the lack of insulin leads to synthesis and storage oftriglycerides in adipose tissue, which prevents weight gain and in turndramatically reduces plasma leptin levels since leptin is synthesized inadipose tissue. These and other Leptin deficiencies, and disease anddisorders that result from such deficiencies, can be treated with leptinreplacement therapy, such as via daily leptin or leptin agonistinjections. The engineered polypeptides described herein can provide amore convenient and advantageous therapeutic treatment of such diseasesand disorders.

Diabetes and Cardiovascular Disease.

Diabetes mellitus is recognized as a complex, chronic disease in which60% to 70% of all case fatalities among diabetic patients are a resultof cardiovascular complications. Diabetes is not only considered acoronary heart disease risk equivalent but is also identified as anindependent predictor of adverse events, including recurrent myocardialinfarction, congestive heart failure, and death following acardiovascular incident. The adoption of tighter glucose control andaggressive treatment for cardiovascular risk factors would be expectedto reduce the risk of coronary heart disease complications and improveoverall survival among diabetic patients. Yet, diabetic patients are twoto three times more likely to experience an acute myocardial infarctionthan non-diabetic patients, and diabetic patients live eight to thirteenyears less than non-diabetic patients.

Understanding the high risk nature of diabetic/acute myocardialinfarction patients, the American College of Cardiology/American HeartAssociation (“ACC/AHA”) clinical practice guidelines for the managementof hospitalized patients with unstable angina or non-ST-elevationmyocardial infarction (collectively referred to as “ACS”) recentlyrecognized that hospitalized diabetic patients are a special populationrequiring aggressive management of hyperglycemia. Specifically, theguidelines state that glucose-lowering therapy for hospitalizeddiabetic/ACS patients should be targeted to achieve preprandial glucoseless than 10 mg/dL, a maximum daily target than 180 mg/dL, and apost-discharge hemoglobin A1c less than 7%.

In a nationwide sample of elderly ACS patients, it was demonstrated thatan increase in 30-day mortality in diabetic patients corresponded withthe patients having higher glucose values upon admission to thehospital. See “Diabetic Coronary Artery Disease & Intervention,”Coronary Therapeutics 2002, Oak Brook, Ill., Sep. 20, 2002. There isincreasing evidence that sustained hyperglycemia rather than transientelevated glucose upon hospital admission is related to serious adverseevents. Although the ideal metric for hyperglycemia and vascular risk inpatients is not readily known, it appears that the mean glucose valueduring hospitalization is most predictive of mortality. In a separatestudy of ACS patients form over forty hospitals in the United States, itwas found that persistent hyperglycemia, as opposed to random glucosevalues upon admission to the hospital, was more predictive ofin-hospital mortality. See Acute Coronary Syndrome Summit: A State ofthe Art Approach, Kansas City, Mo., Sep. 21, 2002. Compared with glucosevalues upon admission, a logistic regression model of glucose controlover the entire hospitalization was most predictive of mortality. Therewas nearly a two-fold increased risk of mortality during hospitalizationfor each 10 mg/dL increase in glucose over 120 mg/dL. In a smallercohort of consecutive diabetic/ACS patients, there was a graded increasein mortality at one year with increasing glucose levels upon hospitaladmission. In the hospital setting, the ACC/AHA guidelines suggestinitiation of aggressive insulin therapy to achieve lower blood glucoseduring hospitalization.

It has been reported that leptin can have direct benefit to treatingdiabetes, particularly in type I diabetes and type II diabetes, with orwithout the presence of obesity, and more particularly in conditions oflow serum leptin. It has been reported that leptin replenishment reducedor prevented hyperinsulinemia, insulin resistance and hyperglycemia invarious animal models of diabetes type 1 and 2 with or without attendantobesity. For example, high leptin plasma levels generated either bypharmacological administration of leptin or with adenoviral gene therapyreduced hyperglycemia and associated increases of plasma glucagon levelsin STZ-induced diabetes, despite persistently low insulin levels.

Lipid Regulation Diseases.

As known in the art, lipodystrophy is characterized by abnormal ordegenerative conditions of the body's adipose tissue. Dyslipidemia is adisruption in the normal lipid component in the blood. It is believedthat prolonged elevation of insulin levels can lead to dyslipidemia.Hyperlipidemia is the presence of raised or abnormal levels of lipidsand/or lipoproteins in the blood. Hypothalamic amenorrhea is a conditionin which menstruation stops for several months due to a probleminvolving the hypothalamus. It has been found that leptin replacementtherapy in women with hypothalamic amenorrhea improves reproductive,thyroid, and growth hormone axes and markers of bone formation withoutcausing adverse effects. See e.g., Oral et al., N Engl J Med. 2004, 351:959-962, 987-997. Fatty liver disease, e.g., nonalcoholic fatty liverdisease (NAFLD) refers to a wide spectrum of liver disease ranging fromsimple fatty liver (steatosis), to nonalcoholic steatohepatitis (NASH),to cirrhosis (irreversible, advanced scarring of the liver). All of thestages of NAFLD have in common the accumulation of fat (fattyinfiltration) in the liver cells (hepatocytes). It is believed thatleptin is one of the key regulators for inflammation and progression offibrosis in various chronic liver diseases including NASH. See e.g.,Ikejima et al., Hepatology Res. 33:151-154.

Additionally, without wishing to be bound by any theory, it is believedthat relative insulin deficiency in type 2 diabetes, glucose toxicity,and increased hepatic free fatty acid burden through elevated deliveryfrom intra-abdominal adipose tissue via the portal vein, are implicatedas possible causes in fatty liver disorders. Indeed, it has beenhypothesized that eating behavior is the key factor driving themetabolic syndrome of obesity with its many corollaries, including NASH.Accordingly, treatments aimed at decreasing food intake and increasingthe number of small meals, as has already been demonstrated in type 2diabetes, may effectively treat and prevent NASH. Drugs that promoteinsulin secretion and weight loss, and delay gastric emptying are alsoeffective at improving glucose tolerance and thus may improve fattyliver with its attendant hyperinsulinemia. Thus, use of a leptin, leptinanalog, e.g., metreleptin, or an active fragment thereof, can be wellsuited as a treatment modality for this condition. Accordingly,engineered polypeptides described herein which include a leptin, leptinanalog or an active fragment thereof, can be useful in the treatment offatty liver disorders.

Alzheimer's Disease.

Alzheimer's disease (AD), as known in the art, is associated withplaques and tangles in the brain which include dysregulation of theA-beta protein. It is believed that brain lipids are intricatelyinvolved in A-beta-related pathogenic pathways, and that an importantmodulator of lipid homeostasis is leptin. Accordingly, leptin canmodulate bidirectional A-beta kinesis, reducing its levelsextracellularly. Indeed, it has been demonstrated that chronicadministration of leptin to AD-transgenic animals reduced the brainA-beta load, underlying its therapeutic potential. See Fewlass et al.,2004, FASEB J., 18:1870-1878. Additionally, type 2 diabetes mellitus andAD share epidemiological and biochemical features in that both arecharacterized by insoluble protein aggregates with a fibrillarconformation-amylin in type 2 DM pancreatic islets, and Aβ in AD brain.Without wishing to be bound by any theory, it is believed that similartoxic mechanisms may characterize type-2 DM and AD. See Lim et al., FEBSLett., 582:2188-2194.

Metabolic Syndrome X.

Metabolic Syndrome X is characterized by insulin resistance,dyslipidemia, hypertension, and visceral distribution of adipose tissue,and plays a pivotal role in the pathophysiology of type 2 diabetes. Ithas also been found to be strongly correlated with NASH, fibrosis, andcirrhosis of the liver. Accordingly, engineered polypeptides describedherein can be useful in the treatment of metabolic syndrome X.

Huntington's Disease.

Huntington's Disease is an autosomal dominant, neurogenerative disease.Features of the disease include motor disturbances, dementia,psychiatric problems, and unintended weight loss. Chimeric polypeptidesdescribed herein can be useful in the treatment of Huntington's Disease.

Accordingly, in one aspect, there is provided a method for treating adisease or disorder in a subject. The subject is in need of treatmentfor the disease or disorder. The disease or disorder can belipodystrophy, dyslipidemia, hyperlipidemia, overweight, obesity,hypothalamic amenorrhea, Alzheimer's disease, leptin deficiency, fattyliver disease or diabetes (including type I and type II). Additionaldiseases and disorders which can be treated by the compounds and methodsdescribed herein include nonalcoholic steatohepatitis (NASH) andnonalcoholic fatty liver disease (NAFLD), metabolic syndrome X andHuntington's Disease. The method of treatment includes administration tothe subject of a engineered polypeptide as described herein in an amounteffective to treatment the disease or disorder. The engineeredpolypeptide will include as HD1a leptin, a leptin fragment or a leptinanalog. Accordingly, the engineered polypeptide can have one of thefollowing structures: ABD-HD1, HD1-ABD, ABD-L1-HD1 or HD1-L1-ABD.

In all of the treatment embodiments described herein, the leptin can behuman leptin or metreleptin. In some embodiments, the leptin analog hasat least 50%, for example, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 98% or even higher, identity with human leptin. In someembodiments, the leptin analog has at least 50%, for example, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or even higher, identitywith mouse leptin. In some embodiments, the leptin analog has at least50%, for example, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%or even higher, identity with rat leptin. In some embodiments, theleptin analog has at least 50%, for example, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 98% or even higher, identity with platypusleptin. In some embodiments, the leptin analog has at least 50%, forexample, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or evenhigher, identity with seal leptin. In some embodiments, the leptinanalog is leptin A100, A300 or A500.

V. Assays

Methods for production and assay of engineered polypeptides describedherein are generally available to the skilled artisan. Further, specificmethods are described herein as well as in the patent publications andother references cited herein, which are incorporated by reference forthis additional purpose.

Food Intake.

Without wishing to be bound by any theory, it is believed that foodintake is useful in the assessment of the utility of a compound asdescribed herein. For example, it is known that a number of metabolicpathologies are related to food intake (e.g., diabetes, obesity).Accordingly, an initial screening can be conducted to determine theextent to which food intake is modulated by administration of compoundsdescribed herein, and a positive initial screening can be useful insubsequent development of a compound.

A variety of food intake assays are available to one of skill in theart. For example, in the so-called “home cage model” of food intake,subjects (e.g., rats) are maintained in their home cage, and food intakealong with total weight of the subject is measured following injectionof test compound. In the so-called “feeding patterns model” of foodintake assay, subjects (e.g., rats) are habituated to a feeding chamberand to injections prior to testing. After test compound administration,the subjects are immediately placed into the feeding chamber, and foodintake is automatically determined as a function of time (e.g., 1-minintervals). For both tests, the food is standard chow or any of avariety of chows (e.g., high fat) known in the art. In the so-called“mouse food intake” assay, a test compound may be tested for appetitesuppression, or for an effect on body weight gain in diet-inducedobesity (DIO) mice. In a typical mouse food intake assay, femaleNIH/Swiss mice (8-24 weeks old) are group housed with a 12:12 hourlight:dark cycle with lights on at 0600. Water and a standard pelletedmouse chow diet are available ad libitum, except as noted. Animals arefasted starting at approximately 1500 hrs, 1 day prior to experiment.The morning of the experiment, animals are divided into experimentalgroups. In a typical study, n=4 cages with 3 mice/cage. At time=0 min,all animals are given an intraperitoneal injection of vehicle orcompound, typically in an amount ranging from about 10 nmol/kg to 75nmol/kg, and immediately given a pre-weighed amount (10-15 g) of thestandard chow. Food is removed and weighed at various times, typically30, 60, and 120 minutes, to determine the amount of food consumed. Seee.g., Morley et al., 1994, Am. J. Physiol. 267:R178-R184). Food intakeis calculated by subtracting the weight of the food remaining at thee.g. 30, 60, 120, 180 and/or 240 minute time point, from the weight ofthe food provided initially at time=0. Significant treatment effects areidentified by ANOVA (p<0.05). Where a significant difference exists,test means are compared to the control mean using Dunnett's test (Prismv. 2.01, GraphPad Software Inc., San Diego, Calif.). For any testdescribed herein, administration of test compound can be by any means,including injection (e.g., subcutaneous, intraperitoneal, and the like),oral, or other methods of administration known in the art.

In Vitro Assays.

Without wishing to be bound by any theory or mechanism of action, it isbelieved that correlations exist between the results of in vitro (e.g.,receptor) assays, and the utility of agents for the treatment ofmetabolic diseases and disorders. Accordingly, in vitro assays (e.g.,cell based assays) are useful as a screening strategy for potentialmetabolic agents, such as described herein. A variety of in vitro assaysare known in the art, including those described as follows.

Leptin Binding Assay.

Leptin binding can be measured by the potency of a test compound indisplacing ¹²⁵I-recombinant-Leptin (murine) from the surface membraneexpressing chimeric Leptin (Hu)-EPO (Mu) receptor presented by the 32DOBECA cell line (J Biol Chem 1998; 273(29): 18365-18373). Purified cellmembranes can be prepared by homogenization from harvested confluentcell cultures of 32D OBECA cells. Membranes can be incubated with¹²⁵I-rec-Murine-Leptin and increasing concentrations of test compoundfor 3 hours at ambient temperature in 96-well polystyrene plates. Boundand unbound ligand fractions can then be separated by rapid filtrationonto 96-well GF/B plates pre-blocked for at least 60′ in 0.5% PEI(polyethyleneimine). Glass fiber plates can then be dried, scintillantadded, and CPM determined by reading on a multiwell scintillationcounter capable of reading radiolabeled iodine.

Leptin Functional Assay.

Increased levels of phosphorylated STAT5 (Signal Transducer andActivator of Transcription 5) can be measured following treatment of32D-Keptin cells ectopically expressing chimeric Hu-Leptin/Mu-EPOreceptor with a test compound. The 32D-Keptin cells (identical to32D-OBECA cells but maintained in culture with leptin) can be leptinweaned overnight and then treated with test compounds in 96-well platesfor 30 minutes at 37° C. followed by cell extraction. The pSTAT5 levelsin the cell lysates can be determined using the Perkin ElmerAlphaScreen® SureFire® pSTAT5 assay kit in a 384-well format(Proxiplate™ 384 Plus). The efficacy of test compounds can be determinedrelative to the maximal signal in cell lysates from cells treated withHuman leptin.

VI. Pharmaceutical Compositions

In one aspect, there are provided pharmaceutical compositions comprisingcompounds described herein in combination with a pharmaceuticallyacceptable excipient (e.g., carrier). The term “pharmaceuticallyacceptable carrier,” as used herein refers to pharmaceutical excipients,for example, pharmaceutically, physiologically, acceptable organic orinorganic carrier substances suitable for enteral or parenteralapplication that do not deleteriously react with the active agent.Suitable pharmaceutically acceptable carriers include water, saltsolutions (e.g., Ringer's solution and the like), alcohols, oils,gelatins, and carbohydrates such as lactose, amylose or starch, fattyacid esters, hydroxymethycellulose, and polyvinyl pyrrolidine. Suchpreparations can be sterilized and, if desired, mixed with auxiliaryagents such as lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, coloring,and/or aromatic substances and the like that do not deleteriously reactwith the compounds of the invention.

In a further aspect, there is provided a pharmaceutical compositionwhich includes a engineered polypeptide as described herein incombination with a pharmaceutically acceptable excipient. In oneembodiment, the pharmaceutical composition is an oral pharmaceuticalcomposition, as described herein. In some embodiments, thepharmaceutical composition is a long lasting pharmaceutical composition.The term “long lasting” in the context of administration of apharmaceutical composition refers to duration of action. Accordingly, along lasting pharmaceutical composition may be administered at intervalsof, for example, 1 hr, 2 hr, 4 hr, 8 hr, 12 hr, 1 day, 2 days, 3 days, 4days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or even longer.In a preferred embodiment, administration is once a day (i.e., “oncedaily”). In a more preferred embodiments, administration is once a week(i.e., “once weekly”).

A. Methods

The engineered polypeptides described herein can be administered aloneor can be co-administered to a subject. Co-administration is meant toinclude simultaneous or sequential administration of the compoundsindividually or in combination (more than one compound). For example, ithas been found that obesity can be beneficially treated with acombination therapy including a leptin (e.g., metreleptin) and certainother anti-obesity compounds. See e.g., U.S. Published Appl. No.2008/0207512. Accordingly, an engineered polypeptide described hereincomprising an ABD and a leptin could be useful for treatment of obesity.Alternatively, the individual engineered polypeptides having can beco-administered with other anti-obesity agents, such as exenatide orliraglutide.

The preparations can also be co-administered, when desired, with otheractive substances (e.g. to reduce metabolic degradation) as known in theart or other therapeutically active agents.

Amylins.

Amylin is a peptide hormone synthesized by pancreatic β-cells that isco-secreted with insulin in response to nutrient intake. The sequence ofamylin is highly preserved across mammalian species, with structuralsimilarities to calcitonin gene-related peptide (CGRP), the calcitonins,the intermedins, and adrenomedullin, as known in the art. Theglucoregulatory actions of amylin complement those of insulin byregulating the rate of glucose appearance in the circulation viasuppression of nutrient-stimulated glucagon secretion and slowinggastric emptying. In insulin-treated patients with diabetes,pramlintide, a synthetic and equipotent analogue of human amylin,reduces postprandial glucose excursions by suppressing inappropriatelyelevated postprandial glucagon secretion and slowing gastric emptying.The sequences of rat amylin, human amylin and pramlintide follow:

rat amylin: (SEQ ID NO: 108) KCNTATCATQRLANFLVRSSNNLGPVLPPTNVGSNTY;human amylin: (SEQ ID NO: 109) KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY;Pramlintide: (SEQ ID NO: 110) KCNTATCATQRLANFLVHSSNNFGPILPPTNVGSNTY.

Davalintide.

Davalintide, also known as “AC-2307” is a potent amylin agonist usefulin the treatment of a variety of disease indications. See WO 2006/083254and WO 2007/114838, each of which is incorporated by reference herein inits entirety and for all purposes. Davalintide is a chimeric peptide,having an N-terminal loop region of amylin or calcitonin and analogsthereof, an alpha-helical region of at least a portion of analpha-helical region of calcitonin or analogs thereof or analpha-helical region having a portion of an amylin alpha-helical regionand a calcitonin alpha-helical region or analog thereof, and aC-terminal tail region of amylin or calcitonin. The sequences of humancalcitonin, salmon calcitonin and davalintide follow:

human calcitonin: (SEQ ID NO: 111) CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP;salmon calcitonin: (SEQ ID NO: 112) CSNLSTCVLGKLSQELHKLQTYPRTNTGSGTP;davalintide: (SEQ ID NO: 113) KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY.

Without wishing to be bound by any theory, it is believed that amylinsand davalintide, and fragments and analogs thereof, can requireC-terminal amidation to elicit a full biological response. It isunderstood that amylin compounds such as those described herein whichinclude amylins and/or davalintide, and fragment and analogs thereof,can be amidated at the C-terminal.

“Amylin agonist compounds” include native amylin peptides, amylin analogpeptides, and other compounds (e.g., small molecules) that have amylinagonist activity. The “amylin agonist compounds” can be derived fromnatural sources, can be synthetic, or can be derived from recombinantDNA techniques. Amylin agonist compounds have amylin agonist receptorbinding activity and may comprise amino acids (e.g., natural, unnatural,or a combination thereof), peptide mimetics, chemical moieties, and thelike. The skilled artisan will recognize amylin agonist compounds usingamylin receptor binding assays or by measuring amylin agonist activityin soleus muscle assays. In one embodiment, amylin agonist compoundswill have an IC₅₀ of about 200 nM or less, about 100 nM or less, orabout 50 nM or less, in an amylin receptor binding assay, such as thatdescribed herein, in U.S. Pat. No. 5,686,411, and US Publication No.2008/0176804, the disclosures of which are incorporated by referenceherein in their entireties and for all purposes. In one embodiment,amylin agonist compounds will have an EC₅₀ of about 20 nM or less, aboutnM 15 or less, about nM 10 or less, or about nM 5 or less in a soleusmuscle assay, such as that described herein and in U.S. Pat. No.5,686,411. In one embodiment, the amylin agonist compound has at least90% or 100% sequence identity to ^(25,28,29)Pro-human-amylin. In oneembodiment, the amylin agonist compound is a peptide chimera of amylin(e.g., human amylin, rat amylin, and the like) and calcitonin (e.g.,human calcitonin, salmon calcitonin, and the like). Suitable andexemplary amylin agonist compounds are also described in US PublicationNo. 2008/0274952, the disclosure of which is incorporated by referenceherein in its entirety and for all purposes.

By “amylin analog” as used herein is meant an amylin agonist that has atleast 50% sequence identity, preferably at least 70% sequence identity,to a naturally-occurring form of amylin, either rat or human or from anyother species, and is derived from them by modifications includinginsertions, substitutions, extensions, and/or deletions of the referenceamino acid sequence.

The amylin analog sequence can have at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 90%, or 95% amino acid sequence identity with the referenceamylin. In one aspect the analog has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15 or even 16 amino acid substitutions, insertions,extensions, and/or deletions relative to the reference compound. In oneembodiment, the amylin analog may comprise conservative ornon-conservative amino acid substitutions (including non-natural aminoacids and L and D forms). These analogs are preferably peptides, peptidederivatives or peptide mimics. Typical amylin analogs will be peptides,especially of 32-37 amino acids, e.g. 27 to 45, especially 28 to 38, andeven 31-36.

Amylin analogs with identity to rat and human amylin include^(25,28,29)Pro-h-amylin (pramlintide) (SEQ ID NO: 110);des-¹Lys-h-amylin (SEQ ID NO: 161); ²⁵Pro,²⁶Val,^(28,29)Pro-h-amylin(SEQ ID NO: 162); ¹⁸Arg,^(25,28)Pro-h-amylin (SEQ ID NO: 163); des-¹L-hamylin (SEQ ID NO: 164); ¹⁸Arg,^(25,28,29)Pro-h-amylin (SEQ ID NO: 165);des-¹Lys,¹⁸Arg,^(25,28,29)Pro-h-amylin (SEQ ID NO: 166);des-¹,Lys^(25,28,29)Pro-h-amylin (SEQ ID NO: 167);²⁵Pro,²⁶Val,^(28,29)Pro-h-amylin (SEQ ID NO: 168); ²⁸Pro-h-amylin,2,7-Cyclo-[²Asp,⁷Lys]-h-amylin (SEQ ID NO: 169); ²⁻³⁷h-amylin (SEQ IDNO: 170); ¹Ala-h-amylin (SEQ ID NO: 171); ²Ala-h-amylin (SEQ ID NO:172); ^(2,7)Ala-h-amylin (SEQ ID NO: 173); ¹Ser-h-amylin (SEQ ID NO:174); ²⁹Pro-h-amylin (SEQ ID NO: 175); ^(25,28)Pro-h-amylin (SEQ ID NO:176); des-¹Lys,^(25,28)Pro-h-amylin (SEQ ID NO: 177);²³Leu,²⁵Pro,²⁶Val,^(28,29)Pro-h-amylin (SEQ ID NO: 178);²³Leu²⁵Pro²⁶Val²⁸Pro-h-amylin (SEQ ID NO: 179);des-¹Lys,²³Leu,²⁵Pro,²⁶Val,²⁸Pro-h-amylin (SEQ ID NO: 180);¹⁸Arg,²³Leu,²⁵Pro,²⁶Val,²⁸Pro-h-amylin (SEQ ID NO: 181);¹⁸Arg,²³Leu,^(25,28,29)Pro-h-amylin (SEQ ID NO: 182);¹⁸Arg²³Leu,^(25,28)Pro-h-amylin (SEQ ID NO: 183);¹⁷Ile,²³Leu,^(25,28,29)Pro-h-amylin (SEQ ID NO: 184);¹⁷Ile,^(25,28,29)Pro-h-amylin (SEQ ID NO: 185);des-¹Lys,¹⁷¹¹e,²³Leu,^(25,28,29)Pro-h-amylin (SEQ ID NO: 186);¹⁷Ile,¹⁸Arg,²³Leu-h-amylin (SEQ ID NO: 187);¹⁷Ile,¹⁸Arg,²³Leu,²⁶Val,²⁹Pro-h-amylin (SEQ ID NO: 188);¹⁷Ile,¹⁸Arg,²³Leu,²⁵Pro,²⁶Val,^(28,29)Pro-h-amylin (SEQ ID NO: 189);¹³Thr,²¹His,²³Leu,²⁶Ala,²⁸Leu,²⁹Pro,³¹Asp-h-amylin (SEQ ID NO: 190);¹³Thr,²¹His,²³Leu,²⁶Ala,²⁹Pro,³¹Asp-h-amylin (SEQ ID NO: 191);des-¹Lys,¹³Thr,²¹His,²³Leu,²⁶Ala,²⁸Pro,³¹Asp-h-amylin (SEQ ID NO: 192);¹³Thr,¹⁸Arg,²¹His,²³Leu,²⁶Ala,²⁹Pro,³¹Asp-h-amylin (SEQ ID NO: 193);¹³Thr,¹⁸Arg,²¹His,²³Leu,^(28,29)Pro,³¹Asp-h-amylin (SEQ ID NO: 194); and¹³Thr,¹⁸Arg,²¹His,²³Leu,²⁵Pro,²⁶Ala,^(28,29)Pro,³¹Asp-h-amylin (SEQ IDNO: 195).

Suitable and exemplary amylin agonist compounds are also described inPCT Patent Publication WO2010/085700.

Amylin analogs include amino acid sequences of residues 1-37 of Formula(I) following, wherein up to 25% of the amino acids set forth in Formula(I) may be deleted or substituted with a different amino acid:

(SEQ ID NO: 800) X′-Xaa¹-Cys²-Asn³-Thr⁴-Ala⁵-Thr⁶-Cys⁷-Ala⁸-Thr⁹-Gln¹⁰-Arg¹¹-Leu¹²-Ala¹³-Asn¹⁴-Phe¹⁵-Leu¹⁶-Val¹⁷-His¹⁸-Ser¹⁹-Ser²⁰-Xaa²¹-Asn²²-Phe²³-Xaa²⁴-Xaa²⁵-Xaa²⁶-Xaa²⁷-Xaa²⁸-Xaa²⁹-Thr³⁰-Xaa³¹-Val³²-Gly³³-Ser³⁴-Asn³⁵-Thr³⁶-Tyr³⁷-X (I).In Formula (I), X′ is hydrogen, an N-terminal capping group, or a linkerto a duration enhancing moiety. Xaa¹ is Lys or a bond, Xaa²¹ is Lys,Cys, or Asn, Xaa²⁴ is Lys, Cys, or Gly, Xaa²⁵ is Lys, Cys, or Pro, Xaa²⁶is Lys, Cys, or Ile, Xaa²⁷ is Lys, Cys, or Leu, Xaa²⁸ is Lys, Cys, orPro, Xaa²⁹ is Lys, Cys, or Pro and Xaa³¹ is Lys, Cys, or Asn. Furtherregarding Formula (I), the variable X represents a C-terminalfunctionality (e.g., a C-terminal cap). X is substituted orunsubstituted amino, substituted or unsubstituted alkylamino,substituted or unsubstituted dialkylamino, substituted or unsubstitutedcycloalkylamino, substituted or unsubstituted arylamino, substituted orunsubstituted aralkylamino, substituted or unsubstituted alkyloxy,substituted or unsubstituted aryloxy, substituted or unsubstitutedaralkyloxy, or hydroxyl. If the C-terminal of the polypeptide componentwith the sequence of residues 1-37 of Formula (I) is capped with afunctionality X, then X is preferably amine thereby forming a C-terminalamide. In some embodiments, up to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45% or even 50% of the amino acids of residues 1-37 of Formula (I) aredeleted or substituted in a polypeptide component according to Formula(I). In some embodiments, the amylin analog component has 0, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or even 16 amino acidsubstitutions relative to the amino acid sequence set forth in Formula(I). In some embodiments, the amylin analog has a sequence which has adefined sequence identity with respect to the residues 1-37 of the aminoacid sequence according to Formula (I). In some embodiments, thesequence identity between an amylin analgo described herein and residues1-37 of Formula (I) is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%or even higher. In some embodiments, up to 50%, 45%, 40%, 35%, 30%, 25%,20%, 15%, 10%, 5% or even less of the amino acids set forth in residues1-37 of Formula (I) may be deleted or substituted with a different aminoacid. In some embodiments, the sequence identity is within the range75%-100%. In some embodiments, the sequence identity is within the range75%-90%. In some embodiments, the sequence identity is within the range80%-90%. In some embodiments, the sequence identity is at least 75%. Insome embodiments, the amylin analog has the sequence of residues 1-37 ofFormula (I).

In some embodiments, amylin analogs including those of Formula (I), formthe basis of a polypeptide component to which one or more durationenhancing moieties are linked, optionally through a linker, to form anamylin polypeptide conjugate. Thus, the polypeptide component serves asa template (“polypeptide template”) to which is attached, preferably bycovalent attachment, one or more duration enhancing moieties. Linkage ofthe duration enhancing moiety to the polypeptide component can bethrough a linker as described herein. Alternatively, linkage of theduration enhancing moiety to the polypeptide component can be via adirect covalent bond. The duration enhancing moiety can be a watersoluble polymer as described herein. In some embodiments, a plurality ofduration enhancing moieties are attached to the polypeptide component,in which case each linker to each duration enhancing moiety isindependently selected from the linkers described herein.

Amylin analogs useful as polypeptide components described hereininclude, but are not limited to, the compounds set forth in residues1-37 of Formula (I) provided in Table 3 below. Unless indicated to thecontrary, all peptides described herein, including peptides having anexpressly provided sequence, are contemplated in both free carboxylateand amidated forms.

TABLE 3 Component polypeptides useful in the compounds described herein.Cmpd Description (sequence) 101KCNTATCATQRLANFLVRSSNNLGPVLPPTNVGSNTY-NH₂ (SEQ ID NO: 160) 102CNTATCATQRLANFLVRSSNNLGPVLPPTNVGSNTY-NH₂ (SEQ ID NO: 801)([desLys¹]-Cmpd 101) 103KCNTATCATQRLANFLVRSSKNLGPVLPPTNVGSNTY-NH₂ (SEQ ID NO: 802) 104CNTATCATQRLANFLVRSSKNLGPVLPPTNVGSNTY-NH₂ (SEQ ID NO: 803)([desLys¹]-Cmpd 103) 105KCNTATCATQRLANFLVRSSNNLGPKLPPTNVGSNTY-NH₂ (SEQ ID NO: 804) 106CNTATCATQRLANFLVRSSNNLGPKLPPTNVGSNTY-NH₂ (SEQ ID NO: 805)([desLys¹]-Cmpd 105) 107KCNTATCATQRLANFLVRSSNNLGPVLPPTKVGSNTY-NH₂ (SEQ ID NO: 806) 108CNTATCATQRLANFLVRSSNNLGPVLPPTKVGSNTY-NH₂ (SEQ ID NO: 807)([desLys¹]-Cmpd 107) 109KCNTATCATQRLANFLVHSSNNFGPILPPTNVGSNTY-NH₂ (SEQ ID NO: 808) 110CNTATCATQRLANFLVHSSNNFGPILPPTNVGSNTY-NH₂ (SEQ ID NO: 809)([desLys¹]-Cmpd 109) 111CNTATCATQRLANFLVHSSKNFGPILPPTNVGSNTY-NH₂ (SEQ ID NO: 810) 112CNTATCATQRLANFLVHSSNNFGPKLPPTNVGSNTY-NH₂ (SEQ ID NO: 811) 113CNTATCATQRLANFLVHSSNNFGPILPPTKVGSNTY-NH₂ (SEQ ID NO: 812) 114CNTATCATQRLANFLVHSSNNFKPILPPTNVGSNTY-NH₂ (SEQ ID NO: 813) 115CNTATCATQRLANFLVHSSNNFGKILPPTNVGSNTY-NH₂ (SEQ ID NO: 814) 116CNTATCATQRLANFLVHSSNNFGPIKPPTNVGSNTY-NH₂ (SEQ ID NO: 815) 117CNTATCATQRLANFLVHSSNNFGPILKPTNVGSNTY-NH₂ (SEQ ID NO: 816) 118CNTATCATQRLANFLVHSSNNFGPILPKTNVGSNTY-NH₂ (SEQ ID NO: 817)

The terms “linker” and the like, in the context of attachment ofduration enhancing moieties to a polypeptide component in an amylinpolypeptide conjugate described herein, means a divalent species (-L-)covalently bonded in turn to a polypeptide component having a valencyavailable for bonding and to a duration enhancing moiety having avalency available for bonding. The available bonding site on thepolypeptide component is conveniently a side chain residue (e.g.,lysine, cysteine, aspartic acid, and homologs thereof). In someembodiments, the available bonding site on the polypeptide component isthe side chain of a lysine or a cysteine residue. In some embodiments,the available bonding site on the polypeptide component is theN-terminal amine. In some embodiments, the available bonding site on thepolypeptide component is the C-terminal carboxyl. In some embodiments,the available bonding site on the polypeptide component is a backboneatom thereof. As used herein, the term “linking amino acid residue”means an amino acid within residues 1-37 of Formula (I) to which aduration enhancing moiety is attached, optionally through a linker.

In some embodiments, compounds are provided having a linker covalentlylinking a polypeptide component with a duration enhancing moiety. Thelinker is optional; i.e., any linker may simply be a bond. In someembodiments, the linker is attached at a side chain of the polypeptidecomponent. In some embodiments, the linker is attached to a backboneatom of the polypeptide component.

In another aspect, there is provided an amylin polypeptide conjugate,which is a derivative of pramlintide with SEQ ID NO:110 or an analogthereof, wherein the amino acid residue in position 1 is absent (i.e.,des-Lys) and an amino acid residue in position 2 to 37 has beensubstituted with a lysine residue or cysteine residue and wherein saidlysine residue or cysteine residue is linked to a polyethylene glycolpolymer, optionally via a linker, wherein the amino acid numberingconforms with the amino acid number in SEQ ID NO:110.

In another aspect, the invention relates to an amylin polypeptideconjugate, which is a derivative of pramlintide with SEQ ID NO:110 or ananalog thereof, wherein the amino acid residue in position 1 is absent(i.e., des-Lys) and wherein an amino acid residue in any one of position2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 31, 32, 33, 34, 35, 36, or 37 issubstituted with a lysine residue and wherein said lysine residue islinked to a polyethylene glycol polymer, optionally via a linker.

In another aspect, the invention relates to an amylin polypeptideconjugate, which is a derivative of pramlintide with SEQ ID NO:110 or ananalog thereof, wherein the amino acid residue in position 1 is absent(i.e., des-Lys) and wherein an amino acid residue in any one of position21, 24-29, or 31 is substituted with a lysine residue and wherein saidlysine residue is linked to a polyethylene glycol polymer, optionallyvia a linker.

In another aspect, the invention relates to an amylin polypeptideconjugate, which is a derivative of pramlintide with SEQ ID NO:110 or ananalog thereof, wherein the amino acid residue in position 1 is absent(i.e., des-Lys) and wherein an amino acid residue in position 21 issubstituted with a lysine residue and wherein said lysine residue islinked to a polyethylene glycol polymer, optionally via a linker.

In another aspect, the invention relates to an amylin polypeptideconjugate, which is a derivative of pramlintide with SEQ ID NO:110 or ananalog thereof, wherein the amino acid residue in position 1 is absent(i.e., des-Lys) and wherein an amino acid residue in position 24 issubstituted with a lysine residue and wherein said lysine residue islinked to a polyethylene glycol polymer, optionally via a linker.

In another aspect, the invention relates to an amylin polypeptideconjugate, which is a derivative of pramlintide with SEQ ID NO:110 or ananalog thereof, wherein the amino acid residue in position 1 is absent(i.e., des-Lys) and wherein an amino acid residue in position 25 issubstituted with a lysine residue and wherein said lysine residue islinked to a polyethylene glycol polymer, optionally via a linker.

In another aspect, the invention relates to an amylin polypeptideconjugate, which is a derivative of pramlintide with SEQ ID NO:110 or ananalog thereof, wherein the amino acid residue in position 1 is absent(i.e., des-Lys) and wherein an amino acid residue in position 26 issubstituted with a lysine residue and wherein said lysine residue islinked to a polyethylene glycol polymer, optionally via a linker.

In another aspect, the invention relates to an amylin polypeptideconjugate, which is a derivative of pramlintide with SEQ ID NO:110 or ananalog thereof, wherein the amino acid residue in position 1 is absent(i.e., des-Lys) and wherein an amino acid residue in position 27 issubstituted with a lysine residue and wherein said lysine residue islinked to a polyethylene glycol polymer, optionally via a linker.

In another aspect, the invention relates to an amylin polypeptideconjugate, which is a derivative of pramlintide with SEQ ID NO:110 or ananalog thereof, wherein the amino acid residue in position 1 is absent(i.e., des-Lys) and wherein an amino acid residue in position 28 issubstituted with a lysine residue and wherein said lysine residue islinked to a polyethylene glycol polymer, optionally via a linker.

In another aspect, the invention relates to an amylin polypeptideconjugate, which is a derivative of pramlintide with SEQ ID NO:110 or ananalog thereof, wherein the amino acid residue in position 1 is absent(i.e., des-Lys) and wherein an amino acid residue in position 29 issubstituted with a lysine residue and wherein said lysine residue islinked to a polyethylene glycol polymer, optionally via a linker.

In another aspect, the invention relates to an amylin polypeptideconjugate, which is a derivative of pramlintide with SEQ ID NO:110 or ananalog thereof, wherein the amino acid residue in position 1 is absent(i.e., des-Lys) and wherein an amino acid residue in position 31 issubstituted with a lysine residue and wherein said lysine residue islinked to a polyethylene glycol polymer, optionally via a linker.

In some embodiments, the duration enhancing moiety is a water-solublepolymer. A “water soluble polymer” means a polymer which is sufficientlysoluble in water under physiologic conditions of e.g., temperature,ionic concentration and the like, as known in the art, to be useful forthe methods described herein. A water soluble polymer can increase thesolubility of a peptide or other biomolecule to which such water solublepolymer is attached. Indeed, such attachment has been proposed as ameans for improving the circulating life, water solubility and/orantigenicity of administered proteins, in vivo. See e.g., U.S. Pat. No.4,179,337; U.S. Published Appl. No. 2008/0032408. Many differentwater-soluble polymers and attachment chemistries have been used towardsthis goal, such as polyethylene glycol, copolymers of ethyleneglycol/propylene glycol, carboxymethylcellulose, dextran, polyvinylalcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymersor random copolymers), and the like.

In some embodiments, the linked duration enhancing moiety includes apolyethylene glycol. Polyethylene glycol (“PEG”) has been used inefforts to obtain therapeutically usable polypeptides. See e.g.,Zalipsky, S., 1995, Bioconjugate Chemistry, 6:150-165; Mehvar, R., 2000,J. Pharm. Pharmaceut. Sci., 3:125-136. As appreciated by one of skill inthe art, the PEG backbone [(CH₂CH₂—O—)_(n), n: number of repeatingmonomers] is flexible and amphiphilic. Without wishing to be bound byany theory or mechanism of action, the long, chain-like PEG molecule ormoiety is believed to be heavily hydrated and in rapid motion when in anaqueous medium. This rapid motion is believed to cause the PEG to sweepout a large volume and prevents the approach and interference of othermolecules. As a result, when attached to another chemical entity (suchas a peptide), PEG polymer chains can protect such chemical entity fromimmune response and other clearance mechanisms. As a result, pegylationcan lead to improved drug efficacy and safety by optimizingpharmacokinetics, increasing bioavailability, and decreasingimmunogenicity and dosing frequency. “Pegylation” refers in thecustomary sense to conjugation of a PEG moiety with another compound.For example, attachment of PEG has been shown to protect proteinsagainst proteolysis. See e.g., Blomhoff, H. K. et al., 1983, BiochimBiophys Acta, 757:202-208. Unless expressly indicated to the contrary,the terms “PEG,” “polyethylene glycol polymer” and the like refer topolyethylene glycol polymer and derivatives thereof, includingmethoxy-PEG (mPEG).

A variety of means have been used to attach polymer moieties such as PEGand related polymers to reactive groups found on the protein. See e.g.,U.S. Pat. No. 4,179,337; U.S. Pat. No. 4,002,531; Abuchowski et al.,1981, in “Enzymes as Drugs,” J. S. Holcerberg and J. Roberts, (Eds.),pp. 367-383; Zalipsky, S., 1995, Bioconjugate Chemistry, 6:150-165. Theuse of PEG and other polymers to modify proteins has been discussed. Seee.g., Cheng, T.-L. et al., 1999m, Bioconjugate Chem., 10:520-528;Belcheva, N. et al., 1999, Bioconjugate Chem., 10:932-937; Bettinger, T.et al., 1998, Bioconjugate Chem., 9:842-846; Huang, S.-Y. et al., 1998,Bioconjugate Chem., 9:612-617; Xu, B. et al. 1998, Langmuir,13:2447-2456; Schwarz, J. B. et al., 1999, J. Amer. Chem. Soc.,121:2662-2673; Reuter, J. D. et al., 1999, Bioconjugate Chem.,10:271-278; Chan, T.-H. et al., 1997, J. Org. Chem., 62:3500-3504.Typical attachment sites in proteins include primary amino groups, suchas those on lysine residues or at the N-terminus, thiol groups, such asthose on cysteine side-chains, and carboxyl groups, such as those onglutamate or aspartate residues or at the C-terminus. Common sites ofattachment are to the sugar residues of glycoproteins, cysteines or tothe N-terminus and lysines of the target polypeptide. The terms“pegylated” and the like refer to covalent attachment of polyethyleneglycol to a polypeptide or other biomolecule, optionally through alinker as described herein and/or as known in the art.

In some embodiments, a PEG moiety in an amylin polypeptide conjugatedescribed herein has a nominal molecular weight within a specifiedrange. As customary in the art, the size of a PEG moiety is indicated byreference to the nominal molecular weight, typically provided inkilodaltons (kD). The molecular weight is calculated in a variety ofways known in the art, including number, weight, viscosity and “Z”average molecular weight. It is understood that polymers, such as PEGand the like, exist as a distribution of molecule weights about anominal average value.

Exemplary of the terminology for molecular weight for PEGs, the term“mPEG40KD” refers to a methoxy polyethylene glycol polymer having anominal molecular weight of 40 kilodaltons. Reference to PEGs of othermolecular weights follows this convention. In some embodiments, the PEGmoiety has a nominal molecular weight in the range 10-100 KD, 20-80 KD,20-60 KD, or 20-40 KD. In some embodiments, the PEG moiety has a nominalmolecular weight of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95 or even 100 KD. Preferably, the PEG moiety has amolecular weight of 20, 25, 30, 40, 60 or 80 KD.

PEG molecules useful for derivatization of polypeptides are typicallyclassified into linear, branched and Warwick (i.e., PolyPEG®) classes ofPEGs, as known in the art. Unless expressly indicated to the contrary,the PEG moieties described herein are linear PEGs. Furthermore, theterms “two arm branched,” “Y-shaped” and the like refer to branched PEGmoieties, as known in the art. The term “Warwick” in the context ofPEGs, also known as “comb” or “comb-type” PEGs, refers to a variety ofmulti-arm PEGs attached to a backbone, typically poly(methacrylate), asknown in the art. Regarding nomenclature including conventions employedin the table provided herein, absent indication to the contrary a PEGmoiety is attached to the backbone of the peptide. For example, Cmpd 119is the result of the conjugation of mPEG40KD to the N-terminal nitrogenof Cmpd 101. Similarly, Cmpd 120 is the result of conjugation ofmPEG40KD to the N-terminal nitrogen of Cmpd 102. Standard single letterabbreviations for amino acids can be used, as can standard three-letterabbreviations. For example, Cmpd 124 is an analog of Cmpd 110 whereinthe residue at position 26 of Cmpd 109 is substituted for lysine, andthe pendant amine functionality of lysine 26 (i.e., K²⁶) is conjugatedwith a PEG40KD moiety. Exemplary compounds are provided in Table 4below.

TABLE 4 Pegylated compounds Cmpd SEQ ID NO: Description 119 196mPEG40KD-Cmpd 101 120 197 mPEG40KD-Cmpd 102 121 198[K²¹(mPEG40KD)]-Cmpd 103 122 199 [K²¹(mPEG40KD)]-Cmpd 104 123 200[K²⁶(mPEG40KD)]-Cmpd 105 124 201 [K²⁶(mPEG40KD)]-Cmpd 106 125 202[K³¹(mPEG40KD)]-Cmpd 107 126 203 [K³¹(mPEG40KD)]-Cmpd 108 127 204[K²⁶(Y-shaped-mPEG40KD)]-Cmpd 105 128 205 [K²¹(mPEG40KD)]-Cmpd 111 129206 [K²⁶(mPEG40KD)]-Cmpd 112 130 207 [K³¹(mPEG40KD)]-Cmpd 113 131 208[K²⁶(Y-shaped-mPEG40KD)]-Cmpd 112 132 209 [K²⁴(mPEG40KD)]-Cmpd 114 133210 [K²⁵(mPEG40KD)]-Cmpd 115 134 211 [K²⁷(mPEG40KD)]-Cmpd 116 135 212[K²⁸(mPEG40KD)]-Cmpd 117 136 213 [K²⁹(mPEG40KD)]-Cmpd 118

Amylins and amylin analogs to which a chemical moiety is attached areamylin derivatives. Amylin derivatives may constitute amylins to which achemical modification has been made of one or more of its amino acidside groups, α-carbon atoms, terminal amino group, or terminalcarboxylic acid group. A chemical modification includes, but is notlimited to, attaching one or more chemical moieties, creating new bonds,and removing one or more chemical moieties. Modifications at amino acidside groups include, without limitation, alkylation, acylation, esterformation, amide formation, maleimide coupling, acylation of lysine8-amino groups, N-alkylation of arginine, histidine, or lysine,alkylation of glutamic or aspartic carboxylic acid groups, anddeamidation of glutamine or asparagine. Modifications of the terminalamino include, without limitation, the desamino, N-lower alkyl,N-di-lower alkyl, and N-acyl modifications. Modifications of theterminal amino include, without limitation, the desamino, N-lower alkyl,N-di-lower alkyl, and N-acyl modifications, such as alkylacyls, branchedalkylacyls, alkylaryl-acyls. Modifications of the terminal carboxy groupinclude, without limitation, the amide, lower alkyl amide, dialkylamide, arylamide, alkylarylamide and lower alkyl ester modifications.Lower alkyl is C₁-C₄ alkyl. Furthermore, one or more side groups, orterminal groups, may be protected by protective groups known to theordinarily-skilled synthetic chemist. The α-carbon of an amino acid maybe mono- or dimethylated.

Amylin derivatives include amylins and amylin analogs conjugated to oneor more water soluble polymer molecules, such as polyethylene glycol(“PEG”), as described above, or fatty acid chains of various lengths(e.g., stearyl, palmitoyl, octanoyl), by the addition of polyaminoacids, such as poly-his, poly-arg, poly-lys, and poly-ala, or byaddition of small molecule substituents include short alkyls andconstrained alkyls (e.g., branched, cyclic, fused, adamantyl), andaromatic groups. In some embodiments, the water soluble polymermolecules will have a molecular weight ranging from about 500 Daltons toabout 60,000 Daltons. See, e.g., PCT Patent Publications WO 2007/104789,WO 2009/034119, and WO 2010/046357 for amylin derivatives suitable foruse as anti-obesity agents in combination with the engineeredpolypeptides of the invention.

Such polymer-conjugations may occur singularly at the N- or C-terminusor at the side chains of amino acid residues within the sequence of anamylin or amylin analog as disclosed herein. Alternatively, there may bemultiple sites of derivatization along the amino acid sequence of suchan amylin or amylin analog. Substitution of one or more amino acids withlysine, aspartic acid, glutamic acid, or cysteine may provide additionalsites for derivatization. In some embodiments, an amylin or amylinanalog may be conjugated to one, two, or three polymer molecules.

In some embodiments, the water soluble polymer molecules are linked toan amino, carboxyl, or thiol group, and may be linked by N or C termini,or at the side chains of lysine, aspartic acid, glutamic acid, orcysteine. Alternatively, the water soluble polymer molecules may belinked with diamine and dicarboxylic groups. In some embodiments, anamylin or amylin analog is conjugated to one, two, or three PEGmolecules through an epsilon amino group on a lysine amino acid.

It has been surprisingly discovered that the engineered polypeptides ofthe invention provide beneficial synergistic anti-obesity effects toboth moderately obese (BMI equal to or greater than 30) and severelyobese (BMI equal to or greater than 35) subjects when administered incombination with certain other anti-obesity compounds. As describedpreviously in, e.g., U.S. Published Appl. No. 2008/0207512, it has beenfound that a state of leptin resistance exists in obese subjects. Seealso, e.g., Tenenbaum, D., HHMI Bulletin, pp. 25-27 (March 2003);Chicurel, M., Nature, Vol. 404, pp. 538-540 (2000); Scarpace et al.,Diabetalogia, Vol. 48, pp. 1075-1083 (2005); and Bays et al., ObesityResearch, Vol. 12, (8), pp. 1197-1211 (2004). This leptin resistance,characterized at least in part by the presence of abnormally high serumleptin levels in obese subjects, makes these subjects unable to respondeffectively to leptin, whether endogenous or exogenously administered.It had been previously found that this leptin resistance could beovercome in moderately obese subjects, with a combination therapyincluding a leptin (e.g., metreleptin) and certain other anti-obesitycompounds. See e.g., U.S. Published Appl. No. 2008/0207512. It hasfurther been found that the synergistic anti-obesity effects of theleptin combination therapy are absent in severely obese, high BMIsubjects, presumably due to a severe leptin resistance. The inventorshave surprisingly discovered that the engineered compounds of theinvention are able to overcome even severe leptin resistance whenadministered in combination with certain other anti-obesity compounds.Accordingly, also provided by the invention are methods of treatingobesity and obesity related conditions, disorders, and diseases insubjects, including high BMI subjects, by the administration of at leasttwo different anti-obesity agents, wherein one anti-obesity agent is anengineered polypeptide of the invention and another anti-obesity agentis an amylin, an amylin analog, an amylin agonist, or an amylinderivative (i.e. an amylin agent).

In certain embodiments, the invention provides methods of treatingobesity in subjects in need thereof comprising administration of a firstanti-obesity agent selected from an engineered polypeptide of theinvention in combination with a second anti-obesity agent selected froman amylin, an amylin analog, an amylin agonist, or an amylin derivativewherein the administration of the agents result in a synergistic effectas compared to administration of either agent alone.

In one aspect, methods of the invention provide a synergisticanti-obesity effect among the administered agents. Accordingly, incertain embodiments, administration of a combination of anti-obesityagents results in an effect, e.g., a reduction in nutrient availability,reduction in body weight, reduction in food intake, increase inmetabolism, which is greater than the combination of the results ofadministration of the anti-obesity agent alone (monotherapy).

“Reduced nutrient availability” is meant to include any means by whichthe body reduces the nutrients available to the body to store as fat. Inother words, reducing nutrient availability may be by means thatinclude, but are not limited to, reducing appetite, increasing satiety,affecting food choice/taste aversion, increasing metabolism, and/ordecreasing or inhibiting food absorption. Exemplary mechanisms that maybe affected include delayed gastric emptying or decreased absorption offood in the intestines.

As used herein, a “subject in need thereof” includes subjects who areoverweight, obese, or desirous of losing weight. Obese subjects includeboth the moderately obese, low BMI population and the severely obese,high BMI population. In addition, subjects who are insulin resistant,glucose intolerant, or have any form of diabetes mellitus (e.g., type 1,2 or gestational diabetes) can benefit from the methods of theinvention.

By “metabolic rate” is meant the amount of energy liberated/expended perunit of time. Metabolism per unit time can be estimated by foodconsumption, energy released as heat, or oxygen used in metabolicprocesses. It is generally desirable to have a higher metabolic ratewhen one wants to lose weight. For example, a person with a highmetabolic rate may be able to expend more energy (e.g., the body burnsmore calories) to perform an activity than a person with a low metabolicrate for that activity.

As used herein, “lean mass” or “lean body mass” refers to muscle andbone. Lean body mass does not necessarily indicate fat free mass. Leanbody mass contains a small percentage of fat (roughly 3%) within thecentral nervous system (brain and spinal cord), marrow of bones, andinternal organs. Lean body mass is measured in terms of density. Methodsof measuring fat mass and lean mass include, but are not limited to,underwater weighing, air displacement plethysmograph, x-ray, DEXA scans,MRIs and CT scans. In certain embodiments, fat mass and lean mass ismeasured using underwater weighing as known in the art.

By “fat distribution” is meant the location of fat deposits in the body.Such locations of fat deposition include, for example, subcutaneous,visceral and ectopic fat depots.

By “subcutaneous fat” is meant the deposit of lipids just below theskin's surface. The amount of subcutaneous fat in a subject can bemeasured using any method available for the measurement of subcutaneousfat. Methods of measuring subcutaneous fat are known in the art, forexample, those described in U.S. Pat. No. 6,530,886, the entirety ofwhich is incorporated herein by reference.

By “visceral fat” is meant the deposit of fat as intra-abdominal adiposetissue. Visceral fat surrounds vital organs and can be metabolized bythe liver to produce blood cholesterol. Visceral fat has been associatedwith increased risks of conditions such as polycystic ovary syndrome,metabolic syndrome and cardiovascular diseases.

By “ectopic fat storage” is meant lipid deposits within and aroundtissues and organs that constitute the lean body mass (e.g., skeletalmuscle, heart, liver, pancreas, kidneys, blood vessels). Generally,ectopic fat storage is an accumulation of lipids outside classicaladipose tissue depots in the body.

As used herein, and as well-understood in the art, “treatment” is anapproach for obtaining beneficial or desired results, including clinicalresults. “Treating” or “palliating” a disease, disorder, or conditionmeans that the extent and/or undesirable clinical manifestations of acondition, disorder, or a disease state are lessened and/or time courseof the progression is slowed or lengthened, as compared to not treatingthe disorder. For example, in treating obesity, a decrease in bodyweight, e.g., at least a 5% decrease in body weight, is an example of adesirable treatment result. For purposes of this invention, beneficialor desired clinical results include, but are not limited to, alleviationor amelioration of one or more symptoms, diminishment of extent ofdisease, stabilized (i.e., not worsening) state of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.Further, treating does not necessarily occur by administration of onedose, but often occurs upon administration of a series of doses. Thus, atherapeutically effective amount, an amount sufficient to palliate, oran amount sufficient to treat a disease, disorder, or condition may beadministered in one or more administrations.

As used herein, the term “therapeutically effective amount” means theamount of the active compounds in the composition that will elicit thebiological or medical response in a tissue, system, subject, or humanthat is being sought by the researcher, veterinarian, medical doctor orother clinician, which includes alleviation of the symptoms of thedisorder being treated. The novel methods of treatment of this inventionare for disorders known to those skilled in the art.

As used herein, the term “prophylactically effective amount” means theamount of the active compounds in the composition that will elicit thebiological or medical response in a tissue, system, subject, or humanthat is being sought by the researcher, veterinarian, medical doctor orother clinician, to prevent the onset of obesity or an obesity-relateddisorder, condition or disease in subjects as risk for obesity or theobesity-related disorder, condition or disease.

In another aspect of the present invention, methods for reducing therisk of developing metabolic disorders are provided, where the methodcomprises administering to the subject a combination of anti-obesityagents in effective amounts to reduce the weight of a subject.

In some embodiments of the invention, methods of the invention are usedto increase the metabolic rate in a subject, decrease a reduction in themetabolic rate in a subject, or preserve the metabolic rate in asubject. In certain embodiments, the metabolic rate may involve thepreferential use of the body's fat as an energy source over lean bodytissue. In one aspect, lean body mass is not decreased followingadministration of the combination of anti-obesity agents.

In another aspect, a reduction in the lean body mass is lessened orprevented following administration of the combination of anti-obesityagents. In still another aspect, lean body mass is increased followingadministration of the combination of anti-obesity agents. Suchpreference for fat as the energy source may be determined by comparingthe amount of fatty tissue to lean body tissue, ascertained by measuringtotal body weight and fat content at the beginning and end of thetreatment period. An increase in metabolic rate is a higher level of theuse of calories or another energy source by a subject over a period oftime compared with the level of use of calories or other energy sourceby the subject over another period of time under substantially similaror identical conditions without administration of the combination ofanti-obesity agents. In certain embodiments, the metabolic rate isincreased at least about 5% in a subject, in other embodiments, themetabolic rate is increased at least about 10%, 15%, 20% 25%, 30%, or35% in a subject compared with the level of use of calories or otherenergy source by the subject over another period of time undersubstantially similar or identical conditions without administration ofthe combination of anti-obesity agents. The increase in metabolic ratecan be measured using a respiratory calorimeter, for example. Aneffective amount of the anti-obesity agents as used in these embodimentsis an amount of each agent effective to increase the metabolic rate in asubject when administered in combination compared to a subject notreceiving the agents or only one of the agents.

In another embodiment, a method is provided to reduce a decrease inmetabolic rate in a subject. Such a decrease in metabolic rate can bethe result of any condition or nutritional or physical regimen thatleads to a reduction in metabolic rate, for example, due to a reducedcalorie diet, a restricted diet, or weight loss. A restricted dietincludes allowances or prohibitions, or both on the types of food or theamounts of food or both permitted in a diet, not necessarily based oncalories. For example, as in individual diets, the body compensates witha reduced metabolic rate based on the lower caloric intake. In essence,the body down-regulates the requirement for food, thereby subsisting onless food. As dieting continues, the threshold for caloric intake isreduced. When dieting has ended, the individual typically gains weightwhile eating a normal diet because of the lowered caloric intakethreshold and lower-basal metabolic rate (NIH Technology AssessmentConference Panel (1992) Ann. Intern. Med. 116:942-949; Wadden (1993)Ann. Intern. Med. 119:688-693). In one aspect, a method is provided toreduce the loss of metabolic rate in a subject, where the loss ofmetabolic rate is the result of a reduced calorie diet or weight loss.By using such a method, the subject's reduction in metabolic rate isdecreased by at least about 10%, 15%, 20% 25%, 30%, 35%, 40%, 50%, 60%,70%, 80%, 90%, or 95% in a subject. For such methods, it may bedesirable to administer the combination of anti-obesity agents at thetime the condition or nutritional or physical regimen is initiated whichleads to a loss or reduction in metabolic rate. However, it is alsocontemplated that administration of the agents is commenced before thecondition or nutritional or physical regimen is initiated. In oneinstance, metabolic rate is measured using a respiratory calorimeter. Aneffective amount of the anti-obesity agents of as used in thisembodiment is an amount of each agent effective to decrease thereduction of the metabolic rate in a subject when administered incombination.

In another aspect, methods for reducing metabolic plateaus are provided,where a method comprises administering effective amounts of anti-obesityagents in combination to a subject. In certain embodiments, the subjectis losing weight, or has lost weight, for example, due to a reducedcalorie diet, increased exercise or a combination thereof. By “metabolicplateau” is meant time intervals of steady metabolic rate while the bodyadjusts to changes in caloric or energy input. Changes in caloric inputor expenditure can be the result of, for example, reduced calorie dietsor increased physical activity. Such plateaus can be observed, forexample, during a weight loss regimen when weight loss slows or stops.In certain embodiments, a method of the present invention reduces theduration of a metabolic plateau in a subject compared with the durationof metabolic plateaus in an otherwise identical subject over the sameperiod of time under substantially similar or identical conditionswithout administration of the combination of anti-obesity agents. Inother embodiments, a method of the present invention reduces thefrequency of metabolic plateaus compared with the frequency of metabolicplateaus in an otherwise identical subject over the same period of timeunder substantially similar or identical conditions withoutadministration of the combination of anti-obesity agents. In still otherembodiments, a method of the present invention delays the onset of ametabolic plateau compared with the onset of a metabolic plateau in anotherwise identical subject over the same period of time undersubstantially similar or identical conditions without administration ofthe combination of anti-obesity agents. In certain embodiments,metabolic plateaus are identified by charting periods of reduced or noweight loss. In certain embodiments, at least one metabolic plateau isreduced. In other embodiments, at least two, three, four, five, six,seven, eight, nine, or ten metabolic plateaus are reduced. In anotheraspect, metabolic plateaus are delayed one day as compared to a subjectnot administered the combination of anti-obesity agents under identicalor similar conditions. In other aspects, metabolic plateaus are delayed2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks or 3weeks in a subject.

In yet other embodiments, a method is provided to preserve the metabolicrate in a subject. In certain embodiments, the subject may be at risk oflosing metabolic rate, for example, due to the initiation of a reducedcalorie diet, restricted diet, or anticipated weight loss. Apreservation of metabolic rate is a maintenance of the level of the useof calories or another energy source by a subject over a period of timecompared with the level of use of calories or other energy source by anotherwise identical subject over the same period of time undersubstantially similar or identical conditions without administration ofthe combination of anti-obesity agents. In one aspect, the metabolicrate is maintained within 15% of the subject's metabolic rate prior tothe initiation of the event that results in the decrease in metabolicrate. In other aspects, the metabolic rate is maintained within 10%,within 7%, within 5%, within 3% or less of the subject's metabolic rate.In one aspect, the combination of anti-obesity agents is administered atthe initiation of a reduced calorie diet, restricted diet, or exerciseregimen.

Metabolic rates can be assessed using any method available fordetermining such rates, for example by using a respiratory calorimeter.Such methods and devices for assaying metabolic rates are known in theart and are described, for example, in U.S. Pat. Nos. 4,572,208,4,856,531, 6,468,222, 6,616,615, 6,013,009, and 6,475,158.Alternatively, the metabolic rate of an animal can be assessed bymeasuring the amount of lean tissue versus fatty tissue catabolized bythe animal following the diet period. Thus, total body weight and fatcontent can be measured at the end of the dietary period. In rats, afrequently used method to determine total body fat is to surgicallyremove and weigh the retroperitoneal fat pad, a body of fat located inthe retroperitoneum, the area between the posterior abdominal wall andthe posterior parietal peritoneum. The pad weight is considered to bedirectly related to percent body fat of the animal. Since therelationship between body weight and body fat in rats is linear, obeseanimals have a correspondingly higher percent of body fat andretroperitoneal fat pad weight.

In another aspect of the present invention, methods for reducing fatmass by increasing the metabolic rate in a subject are provided, wherethe methods comprise administering a combination of anti-obesity agentsin amounts effective to reduce fat mass by increasing the subject'smetabolic rate. Fat mass can be expressed as a percentage of the totalbody mass. In some aspects, the fat mass is reduced by at least 1%, atleast 5%, at least 10%, at least 15%, at least 20%, or at least 25% overthe course of treatment. In one aspect, the subject's lean mass is notdecreased over the course of the treatment. In another aspect, thesubject's lean mass is maintained or increased over the course of thetreatment. In another aspect, the subject is on a reduced calorie dietor restricted diet. By “reduced calorie diet” is meant that the subjectis ingesting fewer calories per day than compared to the same subject'snormal diet. In one instance, the subject is consuming at least 50 fewercalories per day. In other instances, the subject is consuming at least100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1000 fewercalories per day.

In certain embodiments of the present invention, a method for alteringthe fat distribution in a subject is provided where the method comprisesadministering a combination of anti-obesity agents in amounts effectiveto alter fat distribution in the subject. In one aspect, the alterationresults from an increased metabolism of visceral or ectopic fat, or bothin the subject. In some embodiments, the method involves the metabolismof visceral or ectopic fat or both at a rate of at least about 5%, 10%,15%, 20%, 25%, 30%, 40%, or 50% greater than for subcutaneous fat. Inone aspect, the methods result in a favorable fat distribution. Incertain embodiments, favorable fat distribution is an increased ratio ofsubcutaneous fat to visceral fat, ectopic fat, or both. In one aspect,the method involves an increase in lean body mass, for example, as aresult of an increase in muscle cell mass.

In other embodiments, methods for reducing the amount of subcutaneousfat in a subject are provided, wherein the method comprisesadministering, to a subject in need thereof, a combination ofanti-obesity agents in amounts effective to reduce the amount ofsubcutaneous fat in the subject. In one instance, the amount ofsubcutaneous fat is reduced in a subject by at least about 5%. In otherinstances, the amount of subcutaneous fat is reduced by at least about10%, 15%, 20%, 25%, 30% 40%, or 50% compared to the subject prior toadministration of the anti-obesity agents.

The methods described herein can be used to reduce the amount ofvisceral fat in a subject. In one instance, the visceral fat is reducedin a subject by at least about 5%. In other instances, the visceral fatis reduced in the subject by at least about 10%, 15%, 20%, 25%, 30% 40%,or 50% compared to the subject prior to administration of thecombination of anti-obesity agents. Visceral fat can be measured throughany means available to determine the amount of visceral fat in asubject. Such methods include, for example, abdominal tomography bymeans of CT scanning and MRI. Other methods for determining visceral fatare described, for example, in U.S. Pat. Nos. 6,864,415, 6,850,797, and6,487,445.

In certain embodiments, a method for preventing the accumulation ofectopic fat or reducing the amount of ectopic fat in a subject isprovided, wherein the method comprises administering, to a subject inneed thereof, a combination of anti-obesity agents in amounts effectiveto prevent accumulation of ectopic fat or to reduce the amount ofectopic fat in the subject. In one instance, the amount of ectopic fatis reduced in a subject by at least about 5% compared to the subjectprior to administration of the combination of anti-obesity agents. Inother instances, the amount of ectopic fat is reduced in a subject by atleast about 10%, or by at least about 15%, 20%, 25%, 30% 40%, or 50%.Alternatively, the amount of ectopic fat is proportionally reduced 5%,10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% incomparison to subcutaneous fat in a subject. Ectopic fat can be measuredin a subject using any method available for measuring ectopic fat.

In other embodiments, methods are provided for producing a morefavorable fat distribution in a subject, where the method comprisesadministering to a subject a combination of anti-obesity agents inamounts effective to produce a favorable fat distribution. In certainembodiments, administration of a combination of anti-obesity agentsreduces the amount of visceral fat or ectopic fat, or both, in asubject. For example, administration of a combination of anti-obesityagents, where at least one anti-obesity agent that acts upon forebrainstructures involved in food intake or body weight modulation or both incombination with administration of at least one anti-obesity agent thatacts upon hindbrain structures involved in food intake or body weightmodulation or both. In certain embodiments, the methods preferentiallyreduce the amount of visceral or ectopic fat, or a combination of both,over the reduction in subcutaneous fat. Such methods result in a higherratio of subcutaneous fat to visceral fat or ectopic fat. Such improvedratios may result in a reduced risk of the development of cardiovasculardiseases, polycystic ovary syndrome, metabolic syndrome, or anycombinations thereof. In certain embodiments, ectopic or visceral fat ismetabolized at a rate 5% greater than subcutaneous fat. In otherembodiments, ectopic or visceral fat is metabolized at a rate at least10% 15%, 20%, 25%, 30% 50%, 60%, 70%, 80%, 90%, or 100% greater thansubcutaneous fat.

In another aspect, methods of the invention include the use of atherapeutically effective amount of a combination of anti-obesity agentsadministered in combination with glucocortico steroids. Glucocorticosteroids have the adverse effect of increasing fat mass and decreasinglean mass. Accordingly, it is contemplated that the anti-obesity agentcombination can be used in conjunction with glucocortico steroids underconditions where glucocortico steroid use is beneficial.

In still another aspect, methods of the invention include the use of atherapeutically effective amount of one anti-obesity agent or acombination of anti-obesity agents administered in combination with atherapeutic agent selected from orlistat, phentermine, topiramate,CONTRAVE, and QNEXA. In some embodiments, the methods of the inventioninclude the use of a therapeutically effective amount of an engineeredpolypeptide of the invention in combination with a therapeutic agentselected from orlistat, phentermine, topiramate, CONTRAVE, and QNEXA. Inother embodiments, the methods of the invention include the use of atherapeutically effective amount of an amylin, an amylin analog, anamylin agonist, or an amylin derivative in combination with atherapeutic agent selected from orlistat, phentermine, topiramate,CONTRAVE, and QNEXA. In other embodiments, the methods of the inventioninclude the use of a therapeutically effective amount of an engineeredcompound of the invention in combination with an amylin, an amylinanalog, an amylin agonist, or an amylin derivative and a therapeuticagent selected from orlistat, phentermine, topiramate, CONTRAVE, andQNEXA.

Also provided are methods to reduce weight in a morbidly obese subjectby first reducing the subject's weight to a level below that of beingmorbidly obese, then administering to the subject a combination ofanti-obesity agents in effective amounts to further reduce the subject'sweight. Methods for reducing a subject's weight to below that of morbidobesity include reducing caloric intake, increasing physical activity,drug therapy, bariatric surgery, such as gastric bypass surgery, or anycombinations of the preceeding methods. In one aspect, administering thecombination of anti-obesity agents further reduces the weight of thesubject. In other embodiments, methods are provided for reducing thebody mass index in a subject having a body mass index of 40 or less byadministering a combination of anti-obesity agents in effective amountsto further reduce the subject's weight.

By reducing weight it is meant that the subject loses a portion ofhis/her total body weight over the course of treatment, whether thecourse of treatment be days, weeks, months or years. Alternatively,reducing weight can be defined as a decrease in proportion of fat massto lean mass (in other words, the subject has lost fat mass, butmaintained or gained lean mass, without necessarily a corresponding lossin total body weight). An effective amount of the anti-obesity agentsadministered in combination in these embodiments is an amount effectiveto reduce a subject's body weight over the course of the treatment, oralternatively an amount effective to reduce the subject's percentage offat mass over the course of the treatment. In certain embodiments, thesubject's body weight is reduced, over the course of treatment, by atleast about 1%, by at least about 5%, by at least about 10%, by at leastabout 15%, or by at least about 20%. Alternatively, the subject'spercentage of fat mass is reduced, over the course of treatment, by atleast 1%, at least 5%, at least 10%, at least 15%, at least 20%, or atleast 25%.

In certain embodiments, methods of reducing weight include improvedadherence to weight maintenance. Without wishing to be bound by anytheory, the restoration of leptin responsiveness achieved by theadministration of anti-obesity agents as described herein overcomes acritical challenge for obese subjects. In prior weight loss methods,leptin levels may still be higher than normal even at a reduced bodyweight, making it difficult for subjects to maintain the weight loss.The methods described herein include not only methods of reducingweight, but also the component of improved adherence to weightmaintenance.

In certain embodiments, methods of reducing nutrient availability, e.g.,reducing weight, in a subject comprise administering to the subject aneffective amount of the anti-obesity agents in a bolus dose one or moretimes a day. A bolus dose is an intermittent dosage of medicine (asopposed to a continuous infusion). A subject can be administered one ormore bolus doses per day. The bolus dose can be the same no matter whenit is administered to the subject, or can be adjusted such that thesubject is administered a larger bolus dose at certain times of the dayas compared to others. Administration of an agent in certainformulations, e.g., sustained-release formulations, a bolus dose can beadministered less frequently, for example, once every three days, onceper week, twice a month, once every month. Furthermore, the time betweenbolus doses is preferably long enough to allow the drug administered inthe previous bolus dose to clear the subject's blood stream.

In other embodiments, methods of reducing nutrient availability, e.g.,reducing weight, in a subject comprise administering to the subject aneffective amount of the anti-obesity agents in continuous doses. Bycontinuous dose it is intended to mean the continuous infusion of thedrug by, for example, intravenous injection or a transdermal patch.Alternatively, a continuous dose can be administered orally in the formof a controlled release capsule or tablet which releases the drug intothe subject's system over a period of time. When administered by acontinuous dose, the drug is released over a period of about 1 hour, insome cases the drug is released over a period of about 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 18, or 24 hours.

By “administered in combination” is meant that the anti-obesity agentsare administered as a single administration, simultaneously as separatedoses, or as sequentially administered. Sequential administration refersto administering one of the anti-obesity agents either before or afteran anti-obesity agent. In certain embodiments, the first anti-obesityagent is administered about 30 minutes before or after the at least oneother anti-obesity agent, in other embodiments about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, or 12 hours before or after the at least one otheranti-obesity agents. Any of the administered anti-obesity agents can beadministered as a bolus dose or as a continuous dose.

Furthermore, in certain embodiments, administration of theweight-inducing agents in combination results in a synergistic effect inany of the aspects of the invention. In addition, in certainembodiments, administration of the weight-inducing agents in combinationresults in a lower dosage requirement for at least one of the agents,with the same effect.

Accordingly, in one embodiment is a method of treating obesity orreducing body weight in a subject in need thereof, comprisingperipherally administering therapeutically effective amounts of at leasttwo different anti-obesity agents, wherein at least one anti-obesityagent is an amylin, an amylin analog, an amylin agonist, or an amylinderivative and at least one anti-obesity agent is an engineeredpolypeptide comprising: an albumin binding domain polypeptide (ABD); anda first peptide hormone domain (HD1) selected from a leptin, a leptinanalog or an active fragment thereof, and the subject reduces bodyweight by least 10%, 12%, 15%, 20%, 30%, 40% or even 50%.

Further embodiments include the following.

Embodiment 1

A method of treating obesity in a subject comprising peripherallyadministering therapeutically effective amounts of at least twodifferent anti-obesity agents, wherein at least one anti-obesity agentis an amylin, an amylin analog, an amylin agonist, or an amylinderivative (i.e. an amylin agent) and at least one anti-obesity agent isan engineered polypeptide comprising: an albumin binding domainpolypeptide (ABD); and a first peptide hormone domain (HD1) selectedfrom a leptin, a leptin analog or an active fragment thereof.

Embodiment 2

A method of reducing body weight in a subject comprising peripherallyadministering therapeutically effective amounts of at least twodifferent anti-obesity agents, wherein at least one anti-obesity agentis an amylin, an amylin analog, an amylin agonist, or an amylinderivative (i.e. an amylin agent) and at least one anti-obesity agent isan engineered polypeptide comprising: an albumin binding domainpolypeptide (ABD); and a first peptide hormone domain (HD1) selectedfrom a leptin, a leptin analog or an active fragment thereof.

Embodiment 3

The method according to any one of embodiments 1 or 2 wherein the atleast one anti-obesity amylin agent is an amylin agonist.

Embodiment 4

The method according to any one of embodiments 1 to 3 wherein the amylinagonist comprises an amylin analog or derivative.

Embodiment 5

The method according to any one of embodiments 1 to 4 wherein the amylinanalog or derivative comprises pramlintide.

Embodiment 6

The method according to any one of embodiments 1 to 5 wherein the amylinanalog or derivative comprises a compound disclosed in Table 4.

Embodiment 7

The method according to any one of embodiments 1 to 6 wherein the amylinanalog or derivative comprises Des-Lys1-[Lys26(mPEG40K)]-Pramlintide(SEQ ID NO: 214).

Embodiment 8

The method according to any one of embodiments 1 to 7 wherein the ABDcomprises any one of the peptides selected from the group consisting of:

(SEQ ID NO: 38) LAEAKVLANRELDKYGVSDFYKSYINRAKTVEGVHTLIGHILAALP,(SEQ ID NO: 39) LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVNALTHHILAALP,(SEQ ID NO: 40) LAEAKVLANRELDKYGVSDYYKNLINRARTVEGVHALIDHILAALP,(SEQ ID NO: 41) LAEAKVLANRELDKYGVSDYYKNIINRAKTVEGVRALKLHILAALP,(SEQ ID NO: 42) LAEAKVLANRELDKYGVSDFYKNLINRAKTVEGVSSLKGHILAALP,(SEQ ID NO: 43) LAEAKVLANRELDKYGVSDYYKNLINKAKTVEGVEALTLHILAALP,(SEQ ID NO: 44) LAEAKVLANRELDKYGVSDFYKNLINRAKTVEGVDALIAHILAALP,(SEQ ID NO: 45) LAEAKVLANRELDKYGVSDFYKSLINRAKTVEGVDALTSHILAALP,(SEQ ID NO: 46) LAEAKVLANRELDKYGVSDFYKNLINRAKTVEGVNSLTSHILAALP,(SEQ ID NO: 47) LAEAKVLANRELDKYGVSDFYKNVINKAKTVEGVEALIADILAALP,(SEQ ID NO: 48) LAEAKVLANRELDKYGVSDYYKNLINKAKTVEGVQALIAHILAALP,(SEQ ID NO: 49) LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP,(SEQ ID NO: 50) LAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALTLHILAALP,(SEQ ID NO: 51) LAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALISEILAALP, and(SEQ ID NO: 52) LAEAKEDAIKELDKYGVSDYYKRLISKAKTVEGVKALISEILAALP.

Embodiment 9

The method according to any one of embodiments 1 to 8 wherein the HD1comprises an amino acid sequence selected from the group consisting of:SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO: 26,SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145,SEQ ID NO:146, SEQ ID NO:664, SEQ ID NO:665, SEQ ID NO:666, SEQ IDNO:667, SEQ ID NO:668, SEQ ID NO:669, SEQ ID NO:670, SEQ ID NO:671, SEQID NO:672, SEQ ID NO:673, SEQ ID NO:674, SEQ ID NO:675, SEQ ID NO:676,and SEQ ID NO:677.

Embodiment 10

The method according to any one of embodiments 1 to 9, wherein the HD1is SEQ ID NO:29.

Embodiment 11

The method according to any one of embodiments 1 to 10, wherein theengineered polypeptide comprises a compound disclosed in Table 2.

Embodiment 12

The method according to any one of embodiments 1 to 11, wherein theengineered polypeptide comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ IDNO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ IDNO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ IDNO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ IDNO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ IDNO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85 SEQ IDNO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ IDNO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ IDNO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ IDNO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQID NO:106, and SEQ ID NO:107.

Embodiment 13

The method according to any one of embodiments 1 to 12, wherein theengineered polypeptide comprises an amino acid sequence of SEQ ID NO:54.

Embodiment 14

The method according to any one of embodiments 1 to 12, wherein theengineered polypeptide comprises an amino acid sequence of SEQ ID NO:61.

Embodiment 15

The method according to any one of embodiments 1 to 14 wherein theeffective amount of the amylin agent and the effective amount of theengineered polypeptide comprises an amount such that a greater amount ofweight loss is achieved when the amylin agent is administered incombination with the engineered polypeptide to said subject than theamount of weight loss achieved when either agent is administered alone.

Embodiment 16

The method according to any one of embodiments 1 to 15 wherein the twoagents are administered at the same time.

Embodiment 17

The method according to any one of embodiments 1 to 16 wherein the twoagents are mixed together.

Embodiment 18

The method according to any one of embodiments 1 to 17 wherein thesubject's BMI is greater than 25.

Embodiment 19

The method according to any one of embodiments 1 to 18 wherein thesubject's BMI is 25 to 35.

Embodiment 20

The method according to any one of embodiments 1 to 19, wherein thesubject's BMI is 25 to 40.

Embodiment 21

The method according to any one of embodiments 1 to 20, wherein thesubject's BMI is 25 to 45.

Embodiment 22

The method according to any one of embodiments 1 to 21, wherein thesubject's BMI is 35 to 45.

Embodiment 23

The method according to any one of embodiments 1 to 22, wherein thesubject's BMI is reduced to less than 30.

Embodiment 24

The method according to any one of embodiments 1 to 23, wherein thesubject's BMI is reduced to less than 25.

Embodiment 25

The method according to any one of embodiments 1 to 24, wherein thesubject's BMI is reduced to normal.

Embodiment 26

The method according to any one of embodiments 1 to 25, wherein weightloss is achieved within 4 weeks of treatment.

Embodiment 27

The method according to any one of embodiments 1 to 26, wherein weightloss is achieved within 8 weeks of treatment.

Embodiment 28

The method according to any one of embodiments 1 to 27, wherein weightloss is achieved within 12 weeks of treatment.

Embodiment 29

The method according to any one of embodiments 1 to 28, wherein weightloss is achieved within 20 weeks of treatment.

Embodiment 30

The method according to any one of embodiments 1 to 29, wherein weightloss is achieved within 24 weeks of treatment.

Embodiment 31

The method according to any one of embodiments 1 to 30, wherein thesubject is human.

Embodiment 32

The method according to any one of embodiments 1 to 31, wherein thesubject is an obese human.

Embodiment 33

The method according to any one of embodiments 1 to 32, wherein theweight loss is reduced by at least 10%.

Embodiment 34

The method according to any one of embodiments 1 to 33, wherein theweight loss is reduced by at least 12%.

Embodiment 35

The method according to any one of embodiments 1 to 34, wherein theweight loss is reduced by at least 15%.

B. Formulations

The pharmaceutical compounds of the invention may be formulated withpharmaceutically acceptable carriers or diluents as well as any otherknown adjuvants and excipients in accordance with conventionaltechniques such as those disclosed in Remington's PharmaceuticalSciences by E. W. Martin. See also Wang et al. (1988) J. of ParenteralSci. and Tech., Technical Report No. 10, Supp. 42:2 S.

In general, the engineered polypeptides may be formulated into a stable,safe pharmaceutical composition for administration to a patient.Pharmaceutical formulations contemplated for use in the methods of theinvention may comprise approximately 0.01 to 1.0% (w/v), in certaincases 0.05 to 1.0%, of the engineered polypeptide, approximately 0.02 to0.5% (w/v) of an acetate, phosphate, citrate or glutamate bufferallowing a pH of the final composition of from about 3.0 to about 7.0;approximately 1.0 to 10% (w/v) of a carbohydrate or polyhydric alcoholtonicifier and, optionally, approximately 0.005 to 1.0% (w/v) of apreservative selected from the group consisting of m-cresol, benzylalcohol, methyl, ethyl, propyl and butyl parabens and phenol. Such apreservative is generally included if the formulated peptide is to beincluded in a multiple use product.

In particular embodiments, a pharmaceutical formulation of the presentengineered polypeptides may contain a range of concentrations of thecompound(s), e.g., between about 0.01% to about 98% w/w, or betweenabout 1 to about 98% w/w, or preferably between 80% and 90% w/w, orpreferably between about 0.01% to about 50% w/w, or more preferablybetween about 10% to about 25% w/w in these embodiments. A sufficientamount of water for injection may be used to obtain the desiredconcentration of solution.

Additional tonicifying agents such as sodium chloride, as well as otherknown excipients, may also be present, if desired. In some cases, suchexcipients are useful in maintenance of the overall tonicity of thecompound. An excipient may be included in the presently describedformulations at various concentrations. For example, an excipient may beincluded in the concentration range from about 0.02% to about 20% w/w,preferably between about 0.02% and 0.5% w/w, about 0.02% to about 10%w/v, or about 1% to about 20% w/w. In addition, similar to the presentformulations themselves, an excipient may be included in solid(including powdered), liquid, semi-solid or gel form.

The pharmaceutical formulations may be composed in various forms, e.g.,solid, liquid, semisolid or liquid. The term “solid”, as used herein, ismeant to encompass all normal uses of this term including, for example,powders and lyophilized formulations. The presently describedformulations may be lyophilized.

The terms buffer, buffer solution and buffered solution, when used withreference to hydrogen-ion concentration or pH, refer to the ability of asystem, particularly an aqueous solution, to resist a change of pH onadding acid or alkali, or on dilution with a solvent. Characteristic ofbuffered solutions, which undergo small changes of pH on addition ofacid or base, is the presence either of a weak acid and a salt of theweak acid, or a weak base and a salt of the weak base. An example of theformer system is acetic acid and sodium acetate. The change of pH isslight as long as the amount of hydronium or hydroxyl ion added does notexceed the capacity of the buffer system to neutralize it.

As described herein, a variety of liquid vehicles are suitable for usein the formulations of engineered polypeptides, for example, water or anaqueous/organic solvent mixture or suspension.

The stability of a engineered polypeptide formulation for use asdescribed herein is enhanced by maintaining the pH of the formulation ina range determined by methods known in the art. In certain embodiments,the pH of the formulation is maintained in the range of about 3.5 to5.0, or about 3.5 to 6.5, in some embodiments from about 3.7 to 4.3, orabout 3.8 to 4.2. In some embodiments, pH may be about 4.0, about 5.0,about 6.0, about 7.0, about 8.0, about 9.0, or even higher. In someembodiments, pH may be in the physiological range, pH 6-8, preferably pH7-7.6.

In certain embodiments, the buffer with the engineered polypeptide is anacetate buffer (preferably at a final formulation concentration of fromabout 1-5 to about 60 mM), phosphate buffer (preferably at a finalformulation concentration of from about 1-5 to about to about 30 mM) orglutamate buffer (preferably at a final formulation concentration offrom about 1-5 to about to about 60 mM). In some embodiments, the bufferis acetate (preferably at a final formulation concentration of fromabout 5 to about 30 mM).

A stabilizer may be included in the formulations but is not necessarilyneeded. If included, however, a stabilizer useful in the practice of thepresent invention is a carbohydrate or a polyhydric alcohol. A suitablestabilizer useful in the practice of the present invention isapproximately 1.0 to 10% (w/v) of a carbohydrate or polyhydric alcohol.The polyhydric alcohols and carbohydrates share the same feature intheir backbones, i.e., —CHOH—CHOH—, which is responsible for stabilizingthe proteins. The polyhydric alcohols include such compounds assorbitol, mannitol, glycerol, and polyethylene glycols (PEGs). Thesecompounds are straight-chain molecules. The carbohydrates, such asmannose, ribose, sucrose, fructose, trehalose, maltose, inositol, andlactose, on the other hand, are cyclic molecules that may contain a ketoor aldehyde group. These two classes of compounds have been demonstratedto be effective in stabilizing protein against denaturation caused byelevated temperature and by freeze-thaw or freeze-drying processes.Suitable carbohydrates include: galactose, arabinose, lactose or anyother carbohydrate which does not have an adverse affect on a diabeticpatient, i.e., the carbohydrate is not metabolized to form unacceptablylarge concentrations of glucose in the blood. Such carbohydrates arewell known in the art as suitable for diabetics. Sucrose and fructoseare suitable for use with the compound in non-diabetic applications(e.g. treating obesity).

In certain embodiments, if a stabilizer is included, the compound isstabilized with a polyhydric alcohol such as sorbitol, mannitol,inositol, glycerol, xylitol, and polypropylene/ethylene glycolcopolymer, as well as various polyethylene glycols (PEG) of molecularweight 200, 400, 1450, 3350, 4000, 6000, 8000 and even higher). Mannitolis the preferred polyhydric alcohol in some embodiments. Another usefulfeature of the lyophilized formulations of the present invention is themaintenance of the tonicity of the lyophilized formulations describedherein with the same formulation component that serves to maintain theirstability. In some embodiments, mannitol is the preferred polyhydricalcohol used for this purpose.

The United States Pharmacopeia (USP) states that anti-microbial agentsin bacteriostatic or fungistatic concentrations must be added topreparations contained in multiple dose containers. They must be presentin adequate concentration at the time of use to prevent themultiplication of microorganisms inadvertently introduced into thepreparation while withdrawing a portion of the contents with ahypodermic needle and syringe, or using other invasive means fordelivery, such as pen injectors. Antimicrobial agents should beevaluated to ensure compatibility with all other components of theformula, and their activity should be evaluated in the total formula toensure that a particular agent that is effective in one formulation isnot ineffective in another. It is not uncommon to find that a particularantimicrobial agent will be effective in one formulation but noteffective in another formulation.

A preservative is, in the common pharmaceutical sense, a substance thatprevents or inhibits microbial growth and may be added to pharmaceuticalformulations for this purpose to avoid consequent spoilage of theformulation by microorganisms. While the amount of the preservative isnot great, it may nevertheless affect the overall stability of thepeptide.

While the preservative for use in the pharmaceutical compositions canrange from 0.005 to 1.0% (w/v), in some embodiments range for eachpreservative, alone or in combination with others, is: benzyl alcohol(0.1-1.0%), or m-cresol (0.1-0.6%), or phenol (0.1-0.8%) or combinationof methyl (0.05-0.25%) and ethyl or propyl or butyl (0.005%-0.03%)parabens. The parabens are lower alkyl esters of para-hydroxybenzoicacid. A detailed description of each preservative is set forth inRemington's Pharmaceutical Sciences (Id.)

Engineered polypeptides may not have a tendency to adsorb onto the glassin a glass container when in a liquid form, therefore, a surfactant maynot be required to further stabilize the pharmaceutical formulation.However, with regard to compounds which do have such a tendency when inliquid form, a surfactant should be used in their formulation. Theseformulations may then be lyophilized. Surfactants frequently causedenaturation of protein, both of hydrophobic disruption and by saltbridge separation. Relatively low concentrations of surfactant may exerta potent denaturing activity, because of the strong interactions betweensurfactant moieties and the reactive sites on proteins. However,judicious use of this interaction can stabilize proteins againstinterfacial or surface denaturation. Surfactants which could furtherstabilize the engineered polypeptide may optionally be present in therange of about 0.001 to 0.3% (w/v) of the total formulation and includepolysorbate 80 (i.e., polyoxyethylene(20) sorbitan monooleate), CHAPS®(i.e., 3-[(3-cholamidopropyl) dimethylammonio]1-propanesulfonate), Brij®(e.g., Brij® 35, which is (polyoxyethylene (23) lauryl ether),poloxamer, or another non-ionic surfactant.

It may also be desirable to add sodium chloride or other salt to adjustthe tonicity of the pharmaceutical formulation, depending on thetonicifier selected. However, this is optional and depends on theparticular formulation selected. Parenteral formulations preferably maybe isotonic or substantially isotonic.

A preferred vehicle for parenteral products is water. Water of suitablequality for parenteral administration can be prepared either bydistillation or by reverse osmosis. Water for injection is the preferredaqueous vehicle for use in the pharmaceutical formulations.

It is possible that other ingredients may be present in thepharmaceutical formulations. Such additional ingredients may include,e.g., wetting agents, emulsifiers, oils, antioxidants, bulking agents,tonicity modifiers, chelating agents, metal ions, oleaginous vehicles,proteins (e.g., human serum albumin, gelatin or proteins) and azwitterion (e.g., an amino acid such as betaine, taurine, arginine,glycine, lysine and histidine). Additionally, polymer solutions, ormixtures with polymers provide the opportunity for controlled release ofthe peptide. Such additional ingredients, of course, should notadversely affect the overall stability of the pharmaceutical formulationof the present invention.

Containers are also an integral part of the formulation of an injectionand may be considered a component, for there is no container that istotally inert, or does not in some way affect the liquid it contains,particularly if the liquid is aqueous. Therefore, the selection of acontainer for a particular injection must be based on a consideration ofthe composition of the container, as well as of the solution, and thetreatment to which it will be subjected. Adsorption of the peptide tothe glass surface of the vial can also be minimized, if necessary, byuse of borosilicate glass, for example, Wheaton Type I borosilicateglass #33 (Wheaton Type 1-33) or its equivalent (Wheaton Glass Co.).Other vendors of similar borosilicate glass vials and cartridgesacceptable for manufacture include Kimbel Glass Co., West Co., BunderGlas GMBH and Form a Vitrum. The biological and chemical properties ofthe compound may be stabilized by formulation and lyophilization in aWheaton Type 1-33 borosilicate serum vial to a final concentration of0.1 mg/ml and 10 mg/ml of the compound in the presence of 5% mannitol,and 0.02% Tween 80.

For formulations to be delivered by injection, in order to permitintroduction of a needle from a hypodermic syringe into a multiple-dosevial and provide for resealing as soon as the needle is withdrawn, theopen end of each vial is preferably sealed with a rubber stopper closureheld in place by an aluminum band.

Stoppers for glass vials, such as, West 4416/50, 4416/50 (Teflon faced)and 4406/40, Abbott 5139 or any equivalent stopper can be used as theclosure for pharmaceutical for injection. For formulations comprisingpeptidic anti-obesity agents, these stoppers are compatible with thepeptide as well as the other components of the formulation. Theinventors have also discovered that these stoppers pass the stopperintegrity test when tested using patient use patterns, e.g., the stoppercan withstand at least about 100 injections. Alternatively, the peptidecan be lyophilized in to vials, syringes or cartridges for subsequentreconstitution. Liquid formulations of the present invention can befilled into one or two chambered cartridges, or one or two chambersyringes.

Each of the components of the pharmaceutical formulation described aboveis known in the art and is described in PHARMACEUTICAL DOSAGE FORMS:PARENTERAL MEDICATIONS, Vol. 1, 2nd ed., Avis et al. Ed., Mercel Dekker,New York, N.Y. 1992, which is incorporated by reference in its entiretyherein and for all purposes.

The manufacturing process for the above liquid formulations generallyinvolves compounding, sterile filtration and filling steps. Thecompounding procedure involves dissolution of ingredients in a specificorder (preservative followed by stabilizer/tonicity agents, buffers andpeptide) or dissolving at the same time.

Alternative formulations, e.g., non-parenteral, may not requiresterilization. However, if sterilization is desired or necessary, anysuitable sterilization process can be used in developing the peptidepharmaceutical formulation of the present invention. Typicalsterilization processes include filtration, steam (moist heat), dryheat, gases (e.g., ethylene oxide, formaldehyde, chlorine dioxide,propylene oxide, beta-propiolacctone, ozone, chloropicrin, peraceticacid methyl bromide and the like), exposure to a radiation source, andaseptic handling. Filtration is the preferred method of sterilizationfor liquid formulations of the present invention. The sterile filtrationinvolves filtration through 0.45 um and 0.22 um (1 or 2) which may beconnected in series. After filtration, the solution is filled intoappropriate vials or containers.

In certain embodiments, the engineered polypeptides described herein areadministered peripherally to the subjects. In some embodiments, theliquid pharmaceutical formulations of the present invention are intendedfor parenteral administration. Suitable routes of administration includeintramuscular, intravenous, subcutaneous, intradermal, intraarticular,intrathecal and the like. In some embodiments, the subcutaneous route ofadministration is preferred. In certain embodiments, mucosal delivery isalso preferred. These routes include, but are not limited to, oral,nasal, sublingual, pulmonary and buccal routes which may includeadministration of the peptide in liquid, semi-solid or solid form. Forformulations comprising engineered polypeptides, administration viathese routes can require substantially more compound to obtain thedesired biological effects due to decreased bioavailability compared toparenteral delivery. In addition, parenteral controlled release deliverycan be achieved by forming polymeric microcapsules, matrices, solutions,implants and devices and administering them parenterally or by surgicalmeans. Examples of controlled release formulations are described in U.S.Pat. Nos. 6,368,630, 6,379,704, and 5,766,627, which are incorporatedherein by reference. These dosage forms may have a lower bioavailabilitydue to entrapment of some of the peptide in the polymer matrix ordevice. See e.g., U.S. Pat. Nos. 6,379,704, 6,379,703, and 6,296,842,each of which is incorporated herein by reference in its entirety andfor all purposes.

The compounds may be provided in dosage unit form containing an amountof the engineered polypeptide that will be effective in one or multipledoses.

As will be recognized by those in the field, an effective amount of theengineered polypeptide will vary with many factors including the age andweight of the subject, the subject's physical condition, the conditionto be treated, and other factors known in the art. An effective amountof the engineered polypeptides will also vary with the particularcombination administered. As described herein, administration of theengineered polypeptides in combination may allow for a reduced amount ofany of the administered engineered polypeptides to be an effectiveamount.

The long-duration of action of the engineered polypeptide can providethe extended duration of action desired, such as once daily or onceweekly administration. The duration of action can be selected, forexample, by choice of ABD and its affinity for albumin. While notwishing to be bound by theory, it is believed that higher affinity toalbumin will yield longer circulation times providing longer duration ofaction. Either or both pharmacodynamic (therapeutic effects) andpharmacokinetic (drug properties) can be measured over time afterdelivery, such as drug plasma levels, acute or chronic glucose and/orHbA1c lowering, insulin plasma levels, food intake inhibition or weightloss.

C. Effective Dosages

Pharmaceutical compositions provided herein include compositions whereinthe active ingredient is contained in a therapeutically effectiveamount, i.e., in an amount effective to achieve its intended purpose.The actual amount effective for a particular application will depend,inter alia, on the condition being treated. For example, whenadministered in methods to treat diabetes, such compositions willcontain an amount of active ingredient effective to achieve the desiredresult (e.g. decreasing fasting blood glucose in a subject). Whenadministered in methods to treat obesity, such compositions will containan amount of active ingredient effective to achieve the desired result(e.g. decrease the body mass).

The dosage and frequency (single or multiple doses) of compoundadministered can vary depending upon a variety of factors, includingroute of administration; size, age, sex, health, body weight, body massindex, and diet of the recipient; nature and extent of symptoms of thedisease being treated (e.g., the disease responsive to compoundsdescribed herein); presence of other diseases or other health-relatedproblems; kind of concurrent treatment; and complications from anydisease or treatment regimen. Other therapeutic regimens or agents canbe used in conjunction with the methods and compounds of the invention.

Therapeutically effective amounts for use in humans may be determinedfrom animal models. For example, a dose for humans can be formulated toachieve a concentration that has been found to be effective in animals.The dosage in humans can be adjusted by monitoring one or morephysiological parameters, including but not limited to blood sugar andbody mass, and adjusting the dosage upwards or downwards, as describedabove and known in the art.

Dosages may be varied depending upon the requirements of the patient andthe compound being employed. The dose administered to a patient, in thecontext of the present invention, should be sufficient to affect abeneficial therapeutic response in the patient over time. The size ofthe dose also will be determined by the existence, nature, and extent ofany adverse side effects. Generally, treatment is initiated with smallerdosages, which are less than the optimum dose of the compound.Thereafter, the dosage is increased by small increments until theoptimum effect under circumstances is reached. In one embodiment of theinvention, the dosage range is 0.001% to 10% w/v. In another embodiment,the dosage range is 0.1% to 5% w/v.

However, typical doses may contain from a lower limit of about 0.1 mg toan upper limit of about 200 mg of the pharmaceutical compound per day.Also contemplated are other dose ranges such as 1 mg to 100 mg of thecompound per dose, and 3 mg to 70 mg per dose. Typically, the dose ofengineered polypeptides with long duration of action is administered,for example, daily and even once weekly. The doses per day may bedelivered in discrete unit doses, provided continuously in a 24 hourperiod or any portion of that the 24 hours.

Dosage amounts and intervals can be adjusted individually to providelevels of the administered compound effective for the particularclinical indication being treated. This will provide a therapeuticregimen that is commensurate with the severity of the individual'sdisease state.

Utilizing the teachings provided herein, an effective prophylactic ortherapeutic treatment regimen can be planned that does not causesubstantial toxicity and yet is entirely effective to treat the clinicalsymptoms demonstrated by the particular patient. This planning shouldinvolve the careful choice of active compound by considering factorssuch as compound potency, relative bioavailability, patient body weight,presence and severity of adverse side effects, preferred mode ofadministration, and the toxicity profile of the selected agent.

The surprising dose-sparing property of the engineered polypeptidesdescribed herein, along with their surprisingly long plasma half-lifeand duration of pharmacological action, provides for a superiorpharmaceutical agent. The superior properties including dose-sparing,allow for lower dosing, thus less or less severe side-effects andimproved cost of goods, and/or more cost-effective and simplerformulations for once daily or once weekly administration not currentlyachieved by the parent compounds alone.

D. Toxicity

The ratio between toxicity and therapeutic effect for a particularcompound is its therapeutic index and can be expressed as the ratiobetween LD₅₀ (the amount of compound lethal in 50% of the population)and ED₅₀ (the amount of compound effective in 50% of the population).Compounds that exhibit high therapeutic indices are preferred.Therapeutic index data obtained from cell culture assays and/or animalstudies can be used in formulating a range of dosages for use in humans.The dosage of such compounds preferably lies within a range of plasmaconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. See, e.g. Fingl etal., In: THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Ch. 1, p. 1, 1975.The exact formulation, route of administration, and dosage can be chosenby the individual physician in view of the patient's condition and theparticular method in which the compound is used.

Without wishing to be bound by any theory, it is believed thatconjugation of an ABD albumin binding domain with a hormone domain asdescribed herein, can provide decreased immunogenicity as judged by areduction in immune response relative to the hormone domain without ABDconjugation. See e.g., WO 2009/016043, incorporated herein by referencein its entirety and for all purposes.

VII. Examples Example 1 Engineered Polypeptide Recovery

Protein sequences were designed and back translated using commercialsoftware to DNA sequence for cloning into an E. coli expression vector.Sequences were either obtained as oligonucleotides and stitched togetherusing standard PCR amplification techniques, or they were digested fromexisting expression constructs using standard restriction enzymes andthen ligated back together. Sequences expressing the protein of interestwere placed in pET45 with a T7 promoter for inducible expression. Afterconstructs were verified by sequencing, the vector DNA was purified andtransformed into an expression host, typically BL21(DE3). A singlecolony was selected to grow a starter culture in 4 ml LB media for ˜6hrs. Glycerol stocks were prepared by adding 100 ul 80% glycerol to 900ul stock and store at −80 C. Optionally, 500 ul uninduced sample wasretained for gel analysis. A 60 ml culture (magic media) was inoculatedusing 60 ul starter culture in a 125 ml Thompson flask and incubated @30 C overnight. Remove 250 ul sample for analysis. Spin down and freezecell pellet for later processing.

Bacterial cells were harvested and subsequently lysed to isolateinclusion bodies. Since the protein was present in the inclusion bodies,these were solubilized and the protein refolded at 4 C. Proteins werethen separated using size exclusion chromatography until only a singleband remained and endotoxin levels were acceptable for in vivo testing.Analytical HPLC, RP-LC-MS and SDS-PAGE gel were run as quality controlmeasures on the final protein. Protein was distributed to predeterminedaliquots and stored at −80 C.

Typical recoveries of engineered polypeptide for the methods describedherein are provided in Table 5 following. Surprisingly, the recoveriesobserved for the compounds and production methods described above can besignificantly higher than recoveries observed with previously reportedconjugated species, e.g., Fc-leptin and the like. Furthermore, theforeign ABD domain did not adversely affect expression, recovery,re-folding, yield or solubility of the recovered engineeredpolypeptides, particularly for the leptin conjugates despite thegenerally recognized difficulties in recovering and handling leptin.

TABLE 5 Recoveries of engineered polypeptides Cmpd Recovery, mg/50 mLculture 1 26.2 2 53.5 3 11.2 4 33.6

Example 2 Leptin In Vitro Functional Activity

Method.

This assay measures receptor signaling following treatment of cellsexpressing a modified Leptin receptor. Test samples were assumed at 100%purity and re-solvated to 10× assay concentration in stimulation buffer.A total of 90 ul of each 10× compound was transferred into a deep wellpp plate and serially diluted (3-fold series) with Stimulation bufferusing the Perkin Elmer Multiprobe® II and program“MSV_Lep_Func_(—)3-Fold_Dil-Deepwell_(—)96.MPT.” The serially dilutedplate was compounded into the 96-well stimulation plate containing2.5×10̂5 cell pellets of 18 hour leptin-weaned Keptin cells, as known inthe art, using MultiMek test program “MSV_Lep_Func_(—)200 ul_Transfer”that transfers 200 ul of each of the diluted compounds and mixes thecells. At this time, the plate was sealed with an adhesive plate coverand placed at 37 C for 30 minutes to allow for stimulation of pSTAT5.See e.g., Crouse et al., 1998, J. Biol. Chem., 273:18365-18373. Afterincubation, the stimulation plate was centrifuged to re-pellet thecells, the supernatant was removed and the remaining cell pellets werefrozen at −80 C (>30 minutes). Cell lysates were made by the addition of100 ul of 1× lysate to the thawed cell pellets (Perkin Elmer pSTAT5Assay kit) with rotation at ambient RT for 20 minutes. The lysates wereclarified at 2500 rpm for 20 minutes and examined in the pSTAT5 Assaykit as 4 ul/well in a 384-well Proxiplate™ according to manufacturerinstructions. The pSTAT5 signal (RFU) was determined using a PackardFusion α-FP HT plate reader set to Alpha read parameters. Assay wascompleted in 384-well Proxiplate™ plates at 11 μl total volume withvalues representing mean of n=4 assay wells per dose point.

With reference to Table 6 following, Cmpds C1-C6 are exemplary leptins,leptin analogs and leptin derivatives, as described herein.Specifically, Cmpd C1 is SEQ ID NO:20 as described herein. Cmpd C2 isSEQ ID NO:30 (i.e., A200). Cmpd C3 is SEQ ID NO:32, to which a single 20kDa polyethyleneglycol (PEG) moiety has been attached via the cysteineresidue at position 78. Methods for the conjugation of peptides andproteins with PEG as known in the art. Cmpd C4 is a PEGylated derivativeof SEQ ID NO:20, in which a single 20 kDa PEG has been attached via theN-terminus of SEQ ID NO:20. Cmpd C5 is a dual PEGylated PEG derivativeof SEQ ID NO:32, in which a single 20 kDa PEG moiety has been attachedvia the cysteine residue at position 78 and a single 20 kDa PEG moietyhas been attached via the N-terminus. Cmpd C6 is a PEGylated derivativein which a single 40 kDa PEG has been attached via the N-terminus.

Results.

As set forth in Table 6 following, engineered polypeptides describedherein (e.g., Cmpds 1-4) have comparable, and even superior, functionalactivity in the Obeca STAT5 assay, compared with a variety of conjugatedleptins.

TABLE 6 In vitro Functional Activity for leptins EC₅₀ nM (Obeca CmpdMolecule or engineered polypeptide type STAT5 assay) C1 SEQ ID NO: 200.038 C2 SEQ ID NO: 30 0.855 C3 SEQ ID NO: 32 - single 20 kDa PEG via 78C 0.319 C4 SEQ ID NO: 20 - single 20 kDa PEG via 0.275 N-terminus C5 SEQID NO: 32 dual PEGylated (20 kDa 2.262 PEG via 78 C and 20 kDa PEG viaN-terminus) C6 SEQ ID NO: 20 - single PEG40 kDa via 0.355 N-terminus  1SEQ ID NO: 53 0.628  2 SEQ ID NO: 54 0.530  3 SEQ ID NO: 55 0.095  4 SEQID NO: 56 0.103  9 SEQ ID NO: 57 0.185 12 SEQ ID NO: 58 1.052 13 SEQ IDNO: 59 0.116 14 SEQ ID NO: 60 0.406 15 SEQ ID NO: 61 0.427 16 SEQ ID NO:62 0.411 17 SEQ ID NO: 63 0.468 18 SEQ ID NO: 64 0.322

Example 3 Change in Body Weight After Single Administration

Method.

Lean Sprague Dawley rats were maintained on a low fat diet during thestudy. Mean body weight was 319 grams at beginning of study. Animalswere divided into six groups (n=6/group). Each group was assigned toreceive one of the following: vehicle; Cmpd 1 at 2.6 mg/kg in vehicle;Cmpd 2 at 2.7 mg/kg in vehicle; Cmpd 4 at 2.7 mg/kg in vehicle; Cmpd 2at 10 mg/kg in vehicle. Each test animal received a single subcutaneousinjection at time=0. Food intake and change in body weight (% vehiclecorrected) were monitored for 14 days, and the results recorded as shown(FIG. 1A and FIG. 1B). Administered compounds: Vehicle (box); Cmpd 1 at2.6 mg/kg (triangle tip up); Cmpd 2 at 2.7 mg/kg (triangle tip down);Cmpd 4 at 2.7 mg/kg (diamond); Cmpd C2 at 10 mg/kg (circle).

Results.

As depicted in FIG. 1A and FIG. 1B, administration of each engineeredpolypeptide resulted in reduced food intake and body weight. Allcompounds were given at an equimolar dose by total compound weight;compounds were all given at 120 nmol/kg (i.e., Cmpd 1 at 2.6 mg/kg; Cmpd2 and Cmpd 4 at 2.7 mg/kg; Cmpd C2 at 10 mg/kg). It should be noted thatCmpd C2 (i.e., A200) is a dimer of two moieties, each moiety consistingof the FC region of IgG1 fused to human leptin. Cmpd 1 and Cmpd 2 havean activity similar to Cmpd C2 which, because it is a dimer, actuallyhas two leptins per molecule. While the efficacy (lowest body weight)appears similar, it is clear that the trend favors both engineeredpolypeptides over Cmpd C2. When viewed on a per mole of leptin basis,the engineered polypeptides are superior for both inhibition of foodintake and body weight, as Cmpd C2 has 2 moles of leptin for in eachFc-leptin dimeric complex, whereas each mole of ABD-leptin moiety hasonly 1 mole of leptin.

Previous results have shown that one needs approximately 500 ug/kg/dayof an A500 compound to affect 9-10% weight loss at 7 days when given bycontinuous infusion to a lean rat. This results in 2.5 mg of A500 leptincompound at 5 days and 3.5 mg of compound at 7 days. Since an A500compound itself is 16067.68 gm/mol and the molecular weight of Cmpd 2 is˜22,510 gm/mol, one would anticipate needing 1.4× more of the ABD fusionprotein over the 5 days. Instead only 1.08× (2.7 mg/2.5 mg) morecompound was given which indicates a surprising dose-sparing property.

Example 4 Change in Body Weight After Single Administration of Cmpd 2

Method.

Lean Sprague Dawley rats were maintained on a low fat diet during thestudy. Mean body weight was 324 grams at beginning of study. Animalswere divided into four groups (n=6/group). Each group was assigned toreceive one of the following: vehicle; Cmpd 2 at 0.3 mg/kg in vehicle;Cmpd 2 at 1.0 mg/kg in vehicle; Cmpd 2 at 3.0 mg/kg in vehicle. Eachtest animal received a single subcutaneous injection at time=0. Foodintake and change in body weight (% vehicle corrected) were monitoredfor 14 days, and the results recorded as shown (FIG. 2A and FIG. 2B).Administered compounds: Vehicle (box); Cmpd 2 at 0.3 mg/kg (triangle tipup); Cmpd 2 at 1.0 mg/kg (triangle tip down); Cmpd 2 at 3.0 mg/kg(diamond).

Results.

As depicted in FIG. 2A and FIG. 2B, administration at each concentrationof engineered polypeptide Cmpd 2 resulted in reduced food intake andbody weight. A dose response is observed in FIG. 2B.

Example 5 Change in Body Weight After Single Administration of Cmpd C2

Method.

Lean Sprague Dawley rats were maintained on a low fat diet during thestudy. Mean body weight was 324 grams at beginning of study. Animalswere divided into four groups (n=6/group). Each group was assigned toreceive one of the following: vehicle; Cmpd C2 at 1.1 mg/kg in vehicle;Cmpd C2 at 3.3 mg/kg in vehicle; Cmpd C2 at 11.1 mg/kg in vehicle. Eachtest animal received a single subcutaneous injection at time=0. Foodintake and change in body weight (% vehicle corrected) were monitoredfor 14 days, and the results recorded as shown (FIG. 3A and FIG. 3B).Administered compounds: Vehicle (box); Cmpd C2 at 1.1 mg/kg (circle);Cmpd C2 at 3.3 mg/kg (box); Cmpd C2 at 11.1 mg/kg (triangle tip up).

Results.

As depicted in FIG. 3A and FIG. 3B, administration at each concentrationof control Cmpd C2 resulted in reduced food intake and body weight.

Example 6 Change in Body Weight After Single Administration of Cmpd C6

Method.

Lean Sprague Dawley rats were maintained on a low fat diet during thestudy. Mean body weight was 324 grams at beginning of study. Animalswere divided into two groups (n=6/group). Each group was assigned toreceive one of the following: vehicle; Cmpd C6 at 2.2 mg/kg in vehicle.Each test animal received a single subcutaneous injection at time=0.Food intake and change in body weight (% vehicle corrected) weremonitored, and the results recorded as shown (FIG. 4A and FIG. 4B).Administered compounds: Vehicle (box); Cmpd C6 at 2.2 mg/kg (triangletip down).

Results.

As depicted in FIG. 4A and FIG. 4B, administration at each concentrationof control Cmpd C6 resulted in reduced food intake and body weight.

Example 7 Change in Body Weight in DIO Rats

Method.

Diet-induced obese (DIO) prone Sprague Dawley Rats averagingapproximately 500 grams were injected IP with test and control compoundson day 0 and day 7 (n=6 per compound). The test compound was SEQ IDNO:54 given at 1.3 mg/kg/week in vehicle. Body weight and food intakewere measured at multiple points (days 0, 4, 7, 12 and 14 d) during thestudy period. Administered compounds: Vehicle (box); SEQ ID NO:54 at 1.3mg/kg in vehicle (triangle tip up).

Results.

The results, depicted in FIG. 5, demonstrate that IP injection at a onceweekly interval results in a 3% weight loss after 7 days as seenpreviously at this dose. Upon a second injection, the rats continued tolose weight resulting in a cumulative vehicle corrected body weight lossof ˜7-8% at 14 days. In contrast and surprisingly, previous studies withFC-leptin (leptin A200) have only resulted in approximately 4% weightloss at 14 days after a 5 mg/kg/week dose with injections at day 0 andday 7 in a similar DIO model.

Example 8 Detection of Engineered Polypeptides in Plasma

Method.

Diet-induced obese (DIO) Sprague Dawley rats averaging approximately 483grams were split into five groups, two of which were implanted withosmotic pumps. One of the two groups with osmotic pumps received acontinuous subcutaneous infusion (CSI) of vehicle alone; the otherreceived a CSI of SEQ ID NO:33 (i.e., A500) in vehicle at a dose of 250μg/kg/day. The other three groups were treated as follows: one groupreceived once-weekly subcutaneous injections of vehicle alone on days 0,7, 14, and 21 of the study; another group received once-weeklysubcutaneous injections of SEQ ID NO:54 (an ABD-A500 engineeredpolypeptide) at a dose of 1.3 mg/kg in vehicle on days 0, 7, 14, and 21of the study; the remaining group received once-weekly subcutaneousinjections of SEQ ID NO:54 at a dose of 3.0 mg/kg in vehicle on days 0,7, 14, and 21 of the study. Blood samples were taken from each animal onDay 27, which was the day the study was terminated.

Results.

The results, depicted in FIGS. 6A-6B, demonstrate that the once weeklyinjections of SEQ ID NO:54 at 1.3 mg/kg resulted in plasma levels thatwere slightly lower to that achieved by continuous infusion of SEQ IDNO:33, and once weekly injections of SEQ ID NO:54 at 3.0 mg/kg resultedin plasma levels that were significantly greater than that achieved withcontinuous infusion of SEQ ID NO:33 (compare FIG. 6A with FIG. 6B; notedifference in scales of the Y-axis of each panel).

Example 9 Change in Body Weight After Single Administration ofEngineered Polypeptides

Method.

Lean Sprague Dawley rats were maintained on a low fat diet during thestudy. Mean body weight was 330 grams at beginning of study. Each testanimal (n=5/group) received a single subcutaneous injection at time=0.Animals were divided into five groups. Each group was assigned toreceive one of the following: vehicle; SEQ ID NO: 54 in vehicle; SEQ IDNO:56 in vehicle; SEQ ID NO: 58 in vehicle; SEQ ID NO: 59 in vehicle.SEQ ID NOS; 54, 56, 58, and 59 were each delivered at a dose of 120nmol/kg. Percent change in body weight for each group was monitored for14 days, and the results recorded as shown (FIG. 7).

Results.

As depicted in FIG. 7, each group of animals that received a singleinjection of one of the SEQ ID NOS tested exhibited significant andsustained weight loss across the 14-day length of the study relative tothe group that received vehicle alone.

Example 10 Change in Body Weight After Single Administration ofEngineered Polypeptides

Method.

Lean Sprague Dawley rats were maintained on a low fat diet during thestudy. Mean body weight was 330 grams at beginning of study. Animalswere divided into six groups (n=5/group). Each test animal received asingle subcutaneous injection at time=0. Each group was assigned toreceive one of the following: vehicle; SEQ ID NO:54 in vehicle; SEQ IDNO:57 in vehicle; SEQ ID NO:60 in vehicle; SEQ ID NO:61 in vehicle; SEQID NO:62 in vehicle. SEQ ID NOS; 54, 57, 60, 61, and 62 were eachdelivered at a dose of 120 nmol/kg. Percent change in body weight foreach group was monitored for 14 days, and the results recorded as shown(FIG. 8).

Results.

As depicted in FIG. 8, each group of animals that received a singleinjection of one of the SEQ ID NOS tested exhibited significant andsustained reduction in body weight relative to the group that receivedvehicle alone.

Example 11 Change in Body Weight and Food Intake After SingleAdministration of Engineered Polypeptides

Method.

Lean Sprague Dawley rats were maintained on a low fat diet during thestudy. Mean body weight was 317 grams at beginning of study. Each testanimal (n=7/group) received a single subcutaneous injection at time=0.Animals were divided into four groups. Each group was assigned toreceive one of the following: vehicle; SEQ ID NO: 54 in vehicle; SEQ IDNO:63 in vehicle; SEQ ID NO:64 in vehicle. SEQ ID NOS; 54, 63, and 64were each delivered at a dose of 120 nmol/kg. Food intake and percentchange in body weight for each group was monitored for 14 days, and theresults recorded as shown (FIG. 9A and FIG. 9B, respectively).

Results.

As depicted in FIG. 9A and FIG. 9B, each group of animals that receiveda single injection of one of the SEQ ID NOS tested exhibited significantand sustained reduction in food intake (FIG. 9A) and body weight (FIG.9B) relative to vehicle alone.

Example 12 Change in Body Weight After Single Administration ofEngineered Polypeptides

Method.

Lean Sprague Dawley rats were maintained on a low fat diet during thestudy. Mean body weight was 330 g at beginning of study. Animals weredivided into six groups. Each test animal (n=5/group) received a singlesubcutaneous injection at time=0. Each group was assigned to receive oneof the following: vehicle; SEQ ID NO: 54 in vehicle; SEQ ID NO:67 invehicle; SEQ ID NO: 68 in vehicle; and SEQ ID NO:69 in vehicle. SEQ IDNOS; 54, 67, 68, and 69 were each delivered at a dose of 120 nmol/kg.Percent change in body weight for each group was monitored for 10 days,and the results recorded as shown (FIG. 10).

Results.

As depicted in FIG. 10, each group of animals that received a singleinjection of one of the SEQ ID NOS tested exhibited significant andsustained reduction in body weight relative to the group that receivedvehicle alone.

Example 13 Change in Body Weight After Single Administration ofEngineered Polypeptides

Method.

Lean Sprague Dawley rats were maintained on a low fat diet during thestudy. Mean body weight was320 grams at beginning of study. Animals weredivided into six groups. Each test animal (n=5/group) received a singlesubcutaneous injection at time=0. Each group was assigned to receive oneof the following: vehicle; SEQ ID NO: 54 in vehicle; SEQ ID NO:104 invehicle; SEQ ID NO:105 in vehicle; SEQ ID NO:106 in vehicle; and SEQ IDNO:107 in vehicle. SEQ ID NOS; 54, 104, 105, 106, and 107 were eachdelivered at a dose of 120 nmol/kg. Change in body weight for each groupwas monitored for 9 days, and the results recorded as shown (FIG. 11Athrough FIG. 11C).

Results.

As depicted in FIGS. 11A-11C, each group of animals that received asingle injection of one of the SEQ ID NOS tested exhibited significantand sustained reduction in food intake (FIG. 11A) and body weight (FIG.11B and FIG. 11C) relative to vehicle alone.

Example 14 Affinity Determination for Albumin Binding Polypeptides

In this example, Compound 2 and Compound 15 were characterized foraffinity to different variants of albumin.

Material and Methods

All studies were conducted on a BioRad ProteOn XPR36 system using a GLCsensor chip at 25 degrees C. For amine coupling the GLC chip wasactivated for 5 minutes using a 1:1 mixture of sulfo-NHS/EDC diluted30-fold from the initial stock in water as shown below. Each albuminsample was diluted to 25 ug/ml in 10 mM Na Acetate pH 5.0 and injectedfor 5 minutes over separate sensor surfaces. Each surface was thenblocked with 1 M ethanolamine pH 8.5. Each albumin was coupled at adensity of 2000-5000 in resonance units.

The binding of an engineered polypeptide was tested using 5 nM as thehighest concentration in a three-fold dilution series. The runningbuffer contained 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.005%tween-20. All samples were tested using a 3-fold dilution series. Eachconcentration series was tested in duplicate. The dissociation phase forthe highest concentration was monitored for 3 hours.

Results

The relative K_(D) measured for the engineered polypeptides arepresented in Table 7 below. The results show that the albumin bindingpolypeptides associate with serum albumins (SA) with high affinity.

TABLE 7 K_(D) of albumin binding polypeptides to albumin variants RatHuman Monkey Dog Mouse Compound name SA SA SA SA SA Units ABD00239 18 16123 201 1,240 pM (SEQ ID NO: 49) Cmpd 2 33 56 158 358 1,970 Cmpd 15 <212 17 40 277

Example 15 Leptin In Vitro Functional Activity in the Presence ofAlbumin

In this assay, the method described in Example 2 was used, exceptalbumin was added to the stimulation buffer to test leptin function ofCompound 2 in the presence of albumin. The albumins tested included 0.1%or 1% bovine serum albumin (BSA), 1% rat serum albumin (RSA), or 1%human serum albumin (HSA). The control sample was A100 leptin with 0.1%BSA.

Results.

As shown in FIG. 12, there were no effects of 1% Bovine/Rat/HumanAlbumin on the EC50 activity generated by Compound 2 in the LeptinFunction Assay. The results are surprising and show that the therapeuticcompounds are active even when bound to albumin.

Example 16 Prolonged Pharamacokinetic Profiles Delivered by EngineeredPolypeptides Following Subcutaneous Injections in Rats

This study was conducted to evaluate Compound 2 and Compound 15 in ratsby comparing their blood concentration versus time profiles, i.e.pharamacokinetic profiles.

Rats were placed into treatment groups. Compound 2 was administeredsubcutaneously at 30 nmol/kg, 60 nmol/kg, or 120 nmol/kg. Blood sampleswere taken at pre, 12, 24, 48, 96, and 144 hr post-administration fromthe lateral tail vein. The concentration of Compound 2 in plasma wasmeasured by an immunoenzymetric assay method.

Compound 15 was administered subcutaneously at 120 nmol/kg. Bloodsamples were taken at pre, 0.5, 1, 2, 4, 6, 24, 48, 72, 96, 120 and 144hr post-administration from the lateral tail vein. The concentration ofCompound 15 in plasma was measured by an immunoenzymetric assay method.

Both Compound 2 (FIG. 13) and Compound 15 (FIGS. 14A-14B) exhibitedprolonged plasma-versus-time profiles.

Example 17 Effect of Engineered Polypeptides Mediated by LeptinReceptors

Method.

Lean Sprague Dawley rats and ZDF rats were used for this study. ZDF ratshave a mutation (fa) which results in a shortened leptin receptor whichdoes not effectively interact with leptin. Mean body weight was 225grams at beginning of study. Animals were divided into two groups(n=5/group). Each group was assigned to receive one of the following:vehicle; Cmpd 2 at 2.7 mg/kg in vehicle. Each test animal received asingle subcutaneous injection at time=0. Change in body weight (%vehicle corrected) was monitored, and the results recorded as shown(FIG. 15A, FIG. 15B). Administered compounds: Vehicle (filled circle);Cmpd 2 at 2.7 mg/kg (filled square).

Results.

As depicted in FIG. 15A and FIG. 15B, Compound 2 is not efficacious inZDF rats, indicating its effects are mediated via leptin receptors.

Example 18 Dose Sparing with Engineered Polypeptides

This study compared doses of A500 (SEQ ID NO:33) and Compound 2 (SEQ IDNO:54) required to achieve a similar amount of weight loss in leanleptin-sensitive rats. The results are shown in FIG. 16. Compound 2dosed at 120 nmol/kg/week achieves ˜18% vehicle corrected weight loss.To achieve the same amount of weight loss with A500 required a BID doseof 120 nmol/kg/d or 1680 nmol/kg (120 per injection×2 for BID×7 days)over the course of a week. Without wishing to be bound by any theory,this “dose sparing” may be at least partially attributable to theimproved PK profile of Compound 2 over A-500.

Example 19 Solubility of Engineered Polypeptides

As set forth in Table 8 following, engineered polypeptides describedherein have surprisingly high solubility in neutral pH.

Solubility was measured with the following assay: 6-10 mg of purifiedproteins were concentrated at 4° C. with centrifugal filter units(Amicon Ultra-15 or Ultra-4, with 3KDa MW cutoff; Millipore) to a volumeof less than 0.5 ml. They were centrifuged at 14,000 rpm for 10 minutesat 4° C. to remove precipitates and the supernatant was transferred to anew tube. The proteins were allowed to equilibrate overnight at roomtemperature in the dark, then were filtered with 0.22 micron syringefilters (Milex GV; Millipore) to remove precipitates. The absorbance atOD280 was measured with a NanoDrop spectrophotometer and theconcentration was calculated using the protein's theoretical molarextinction coefficient.

TABLE 8 Solubility of Engineered Polypeptides Net Charge at Solubilityin PBS, pH 7.4 Compound pI* pH 7.4* (mg/mL)** A100 6.2 −2.8 2.1ABD1-A100 7.1 −0.7 8 (SEQ ID NO.: 147) A-500 6.2 −2.8 42.9 ABD1-A500 7.1−0.7 10.8 (Cmpd 2) ABD1-HuSeal 10.0 +9.1 >80 (Cmpd 15)

Example 20 Stability of Engineered Polypeptides

As set forth in Table 9 following, engineered polypeptides describedherein are chemically stabile. The compounds were formulated at 1 mg/mLin buffers of different pH. As shown in Table 9, the chimericpolypeptides have good potency (Table 9A) and purity (Table 9B) aftertwo weeks at 40° C., as determined by reverse phase high performanceliquid chromatography (HPLC).

TABLE 9A Potency of Engineered Polypeptides % Potency* by Reversed PhaseHPLC, 14 days at 40° C. PBS, Compound pH 3.0 pH 4.0 pH 5.0 pH 6.0 pH 7.0pH 8.0 pH 9.0 pH 7.4 ABD1-HuSeal 102.7 107.2 104.9 108.5 107.3 100.190.7 104.5 (Cmpd 15) ABD1-A500 95.9 95.9 97.3 91.7 87.3 90.8 72.1 92.0(Cmpd 2) ABD1-A100 72.3 82.0 88.8 86.1 83.3 85.8 69.6 89.2 (SEQ ID NO:147) *Potency = Main peak area/ref std area

TABLE 9B Purity of Engineered Polypeptides % Purity by Reversed PhaseHPLC, 14 days at 40° C. PBS, Compound pH 3.0 pH 4.0 pH 5.0 pH 6.0 pH 7.0pH 8.0 pH 9.0 pH 7.4 ABD1-HuSeal 96.7 98.0 98.5 99.8 97.6 93.8 95.1 97.1ABD1-A500 94.9 96.2 96.8 97.3 96.9 97.5 82.7 97.4 ABD1-A100 70.0 79.485.4 86.5 86.8 86.8 70.9 87.8

Example 21 Stability of Engineered Polypeptides

As set forth in Table 10 following, engineered polypeptides describedherein are chemically stabile. Compound 15 was formulated at threedifferent concentrations in the following buffer: 10 mM glutamic acid,2% glycine, 1% sucrose, 0.01% Tween 20, pH 4.25 and stored at 5° C., 15°C., or 25° C. As shown in Table 10, Compound 15 is chemically stable at10, 20, and 30 mg/mL for at least 1 month at 5-25° C., as determined byHPLC.

TABLE 10 Stability of Engineered Polypeptides Concentration Storage(mg/mL) Condition (° C.) 0 2 4 % Potency by RP-HPLC at Time Point (week)10 5 103.1 103.6 103.1 15 103.1 102.7 102.0 25 103.1 103.8 104.0 20 5102.0 103.6 103.7 15 102.0 103.6 104.1 25 102.0 103.2 103.8 30 5 104.1104.1 103.3 15 104.1 103.7 104.0 25 104.1 102.6 103.4 % Purity bySCX-HPLC at Time Point (week) 10 5 97.6 97.3 97.7 15 97.6 97.6 97.5 2597.6 97.4 96.0 20 5 97.9 97.7 97.6 15 97.9 97.6 97.7 25 97.9 97.4 96.430 5 97.7 97.6 97.7 15 97.7 97.7 97.6 25 97.7 97.3 97.2

Example 22 Stability of Engineered Polypeptides

As set forth in Table 11 following, engineered polypeptides describedherein are physically stabile. Compound 15 was formulated at threedifferent concentrations in the following buffer: 10 mM glutamic acid,2% glycine, 1% sucrose, 0.01% Tween 20, pH 4.25 and stored at 37° C. Asshown in Table 11, Compound 15 is physically stable at 10, 20, and 30mg/mL for at least 1 month, as determined by visual analysis.

TABLE 11 Stability of Engineered Polypeptides Storage Appearance underFiberLite Concentration Condition at Time Point (week) (mg/mL) (° C.) 02 4 8 10 5 Clear Clear Clear Clear 15 Clear Clear Clear Clear 25 ClearClear Clear Clear 20 5 Clear Clear Clear Clear 15 Clear Clear ClearClear 25 Clear Clear Clear Clear 30 5 Clear Clear Clear Clear 15 ClearClear Clear Clear 25 Clear Clear Clear Slightly hazy

Example 23 Stability of Engineered Polypeptides

Engineered polypeptides described herein are physically stable. Table 12shows the results of size exclusion chromatography (SEC) performed onA100, ABD1-HuSeal, and ABD1-A500. The engineered polypeptides showlittle to no self-association to dimer/oligomer, compared to A100.

TABLE 12 Stability of Engineered Polypeptides Compound Pk 1 (%) Pk 2 (%)Pk 3 (%) ABD1-HuSeal 99.22 0.78 n/a ABD1-A500 96.73 3.27 n/a A100 88.2111.15 0.65 Pk1 = Monomer Pk2 = Dimer Pk3 = Oligomer (Trimner/Tetramer)

SEC Method:

Column—Tosoh TSK Gel G3000 SWx17.8 mm×30 cm (#08541)

Mobile Phase—10 mM Na Phosphate, pH 7.4±238 mM NaCl+2.7 mM KCl

Run Time—22 min

Flow Rate—0.8 mL/min

Column Temp—25° C.

Sample Temp—5° C.

Sample load—40 ug

Detection—214 nm

Example 24 Synergy of Amylin and Leptin is Absent in High BMI Subjects

Previous studies had described amylin/leptin synergy in rats weighing500-550 grams. After an inverse relationship of efficacy and BMI wasnoted we assessed the effects of the combination in very obese rats (750grams) and in very obese rats that were food restricted to themoderately obese range (500-550 g) range prior to initiating drugtreatment.

In this study one group of very obese rats (750 g) were allowed to feedad-libitum and were treated with amylin, leptin or the combination ofamylin+leptin. Although amylin was effective, there was no synergyevident with the addition of leptin. A second group of very obese rats(750 g) was calorie restricted down to the 500-550 g range in whichsynergy was previously demonstrated. These animals then beganamylin/leptin treatment and were allowed to feed ad-libitum. FIG. 17shows the results of the study. Rapid weight regain was evident invehicle and leptin monotherapy-treated rats. Some weight maintenance wasachieved with amylin monotherapy. No further weight maintenance wasachieved with the combination. These findings suggest that the lack ofsynergy in “high BMI” rodents cannot simply be rescued by a diet-leadin.

Example 25 Synergy of Engineered Polypeptides with Amylin Agonists

This study examined whether a once weekly administration of PEG-ratamylin (Des-Lys1-[Lys26(mPEG40K)]-Rat Amylin (SEQ ID NO: 148), Compound124) would be sufficient for synergy when co-administered with ABD1-A500(Compound 2). For comparison, ABD1-A500 was also co-administered withinfused rat amylin (FIG. 18A). FIG. 18B shows that although PEG-ratamylin induced weight loss is somewhat slower and of smaller magnitude,the overall amount of weight loss (and synergy) is qualitatively similarto that achieved by infused rat amylin. Amylin was administered at 50μg/kg/d by SC osmotic minipump, PEG-rat amylin was administered at 125nmol/kg once weekly, and ABD1-A500 was administered at 120 nmol/kg onceweekly to male diet induced obese (DIO) Harlan Sprague Dawley (HSD) ratsof 500 g average weight.

Example 26 Synergy of Engineered Polypeptides with Amylin Agonists

This study shows that a once weekly administration of ABD1-HuSeal(Compound 15) is sufficient for synergy when co-adminstered with infusedrat amylin. FIGS. 19A-19B show that the combination of the engineeredpolypeptide and infused amylin resulted in lower food intake (FIG. 19A)and more weight loss (FIG. 19B) than the results observed for each agentalone. ABD1-HuSeal was administered at 120 nmol/kg and amylin wasadministered at 50 μg/kg/d by SC osmotic minipump to male DIO HSD ratsof 500 g average weight.

Example 27 Synergy of Engineered Polypeptides with Amylin Agonists

This study shows that a once weekly administration of ABD1-HuSeal(Compound 15) is sufficient for synergy when co-adminstered with a twiceweekly administration of PEG-rat amylin (Des-Lys1-[Lys26(mPEG40K)]-RatAmylin (SEQ ID NO: 148), Compound 124).

FIG. 20 shows that the combination of the engineered polypeptide andPEG-rat amylin resulted in more weight loss than the results observedfor each agent alone. ABD1-HuSeal was administered at 120 nmol/kg andPEG-amylin was administered at 125 nmol/kg to male DIO HSD rats of 500 gaverage weight.

Example 28 Synergy of Engineered Polypeptides with Amylin Agonists in aHigh BMI Population

FIG. 21A shows the results of previous studies, describing amylin/leptinsynergy in rats weighing 500-550 grams. FIG. 21B shows that this synergyis not observed in a high BMI population of rats (average weight of 700g). FIG. 21C shows once weekly administration of ABD1-A500 (Compound 15)is sufficient for synergy when co-adminstered with a twice weeklyadministration of PEG-rat amylin (Des-Lys1-[Lys26(mPEG40K)]-Rat Amylin(SEQ ID NO: 148), Compound 124) in high BMI rats. ABD1-A500 wasadministered at 120 nmol/kg and PEG-amylin was administered at 125nmol/kg to male DIO HSD rats of 700 g average weight.

Example 29 Synergy of Engineered Polypeptides with Amylin Agonists in aHigh BMI Population

This study shows that a once weekly administration of ABD1-HuSeal(Compound 15) or ABD1-A500 (Compound 2) is sufficient for synergy whenco-adminstered with infused rat amylin to high BMI rats. ABD1-HuSeal(FIG. 22A) or ABD1-A500 (FIG. 22B) was administered at 120 nmol/kg andamylin was administered at 50 μg/kg/d by SC osmotic minipump to male DIOHSD rats of 700 g average weight.

Example 30 Anti-Diabetic Effects of Engineered Polypeptides inNon-Obese, Type 1 Diabetic Mice

The purpose of this study was to evaluate the in vivo effects ofengineered polypeptides on key diabetic and metabolic endpoints in ahigh-dose STZ mouse model of Type 1 diabetes mellitus (T1DM). C57 BL/6male mice were given a single interperitoneal injection of STZ at 200mg/kg to induce Type 1 diabetes. Compounds were administered twice aweek subcutaneously at 120 nmol/kg for two weeks. Measured endpointsincluded HbA1c levels, glucose levels, body weight, and food intake.

FIGS. 23A-23B show that both Compound 15 and Compound 2 normalized bloodglucose in STZ-induced diabetic mice. Both engineered polypeptides alsoreduced Hemoglobin A1c levels, as shown in FIGS. 24A-24B, and reducedbody weight and cumulative food intake, as shown in FIGS. 25A-25B.

In order to ensure that the glucose lowering effects of therapy are notdue to insulin effects, another study was conducted to combine theleptin therapy with a low dose of insulin. Compound 15 was administeredwith or without the addition of a 0.05 U/day dose of insulin in ahigh-dose STZ mouse model of T1DM. C57 BL/6 male mice were given asingle interperitoneal injection of STZ at 175 mg/kg to induce Type 1diabetes. Compounds were administered twice a week subcutaneously at 60nmol/kg for two weeks. Measured endpoints included HbA1c levels, glucoselevels, body weight, and food intake.

FIGS. 26A-26B show a glucose lowering effect potentiated with low doseof insulin in an additive fashion for Compound 15. It also reducedHemoglobin A1c levels, as shown in FIGS. 27A-28B, and reduced bodyweight and cumulative food intake, as shown in FIGS. 28A-28B.

VIII. Embodiments

Additional embodiments of the engineered polypeptides, method of usethereof, and pharmaceuticals compositions described herein follow:

Embodiment 1

An engineered polypeptide comprising: an albumin binding domainpolypeptide (ABD); and a first peptide hormone domain (HD1) selectedfrom a leptin, a leptin analog or an active fragment thereof.

Embodiment 2

The engineered polypeptide according to Embodiment 1, further comprisinga first linker (L1) covalently linked to said HD1.

Embodiment 3

The engineered polypeptide according to Embodiment 1 or 2, wherein saidengineered polypeptide comprises said ABD as an N-terminal moiety andsaid HD1 as a C-terminal moiety.

Embodiment 4

The engineered polypeptide according to Embodiment 1 or 2, wherein saidengineered polypeptide comprises said ABD as a C-terminal moiety andsaid HD1 as an N-terminal moiety.

Embodiment 5

The engineered polypeptide according to Embodiment 3,

comprising the structure: ABD-HD1.

Embodiment 6

The engineered polypeptide according to Embodiment 3,

comprising the structure: ABD-L1-HD1.

Embodiment 7

The engineered polypeptide according to Embodiment 4,

comprising the structure: HD1-ABD.

Embodiment 8

The engineered polypeptide according to Embodiment 4, comprising thestructure: HD1-L1-ABD.

Embodiment 9

The engineered polypeptide according to any one of Embodiments 1 to 8,wherein said HD1 is said a leptin, a leptin analog, a leptin activefragment, or a leptin derivative.

Embodiment 10

The engineered polypeptide according to any one of Embodiments 1 to 9,wherein said HD1 has at least 50% identity with an amino acid sequenceselected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12. SEQ ID NO:13,SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23,SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33,SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146.

Embodiment 11

The engineered polypeptide according to any one of Embodiments 1 to 10,wherein said HD1 has at least 90% identity with an amino acid sequenceselected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.5EQ ID NO:13,SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23,SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33,SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146.

Embodiment 12

The engineered polypeptide according to any one of Embodiments 1 to 11,wherein said HD1 has at least 50% identity with a human leptin.

Embodiment 13

The engineered polypeptide according to any one of Embodiments 1 to 12,wherein said HD1 has at least 90% identity with a human leptin.

Embodiment 14

The engineered polypeptide according to any one of Embodiments 1 to 13,wherein said HD1 has at least 50% identity with SEQ ID NO: 20.

Embodiment 15

The engineered polypeptide according to any one of Embodiments 1 to 14,wherein said HD1 has at least 90% identity with SEQ ID NO: 20.

Embodiment 16

The engineered polypeptide according to any one of Embodiments 1 to 15,wherein said HD1 has at least 50% identity with a platypus leptin.

Embodiment 17

The engineered polypeptide according to any one of Embodiments 1 to 16,wherein said HD1 has at least 50% identity with a seal leptin.

Embodiment 18

The engineered polypeptide according to any one of Embodiments 1 to 17,wherein said HD1 has from 1 to 5 amino acid modifications selectedindependently from any one or combination of an insertion, deletion,addition and substitution.

Embodiment 19

The engineered polypeptide according to any one of Embodiments 1 to 18,wherein said HD1 comprises an amino acid sequence selected from thegroup consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO: 26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO: SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:143, SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146.

Embodiment 20

The engineered polypeptide according to any one of Embodiments 1 to 19,wherein said HD1 comprises an amino acid sequence that is selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO: 26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO: SEQID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33,SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146.

Embodiment 21

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is SEQ ID NO:1.

Embodiment 22

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is SEQ ID NO:2.

Embodiment 23

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is SEQ ID NO:3.

Embodiment 24

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is SEQ ID NO:4.

Embodiment 25

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is: SEQ ID NO:5.

Embodiment 26

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is SEQ ID NO:6.

Embodiment 27

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is SEQ ID NO:7.

Embodiment 28

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is SEQ ID NO:8.

Embodiment 29

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is SEQ ID NO:9.

Embodiment 30

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is SEQ ID NO:10.

Embodiment 31

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is SEQ ID NO:11.

Embodiment 32

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is SEQ ID NO:12.

Embodiment 33

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is SEQ ID NO:13.

Embodiment 34

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is SEQ ID NO:14.

Embodiment 35

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is SEQ ID NO:15.

Embodiment 36

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is SEQ ID NO:16.

Embodiment 37

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is SEQ ID NO:17.

Embodiment 38

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is SEQ ID NO:18.

Embodiment 39

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is SEQ ID NO:19.

Embodiment 40

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is SEQ ID NO:20.

Embodiment 41

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is SEQ ID NO:21.

Embodiment 42

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is SEQ ID NO:22.

Embodiment 43

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is SEQ ID NO:23.

Embodiment 44

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is SEQ ID NO:24.

Embodiment 45

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is SEQ ID NO:25.

Embodiment 46

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is SEQ ID NO: 26.

Embodiment 47

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is SEQ ID NO:27.

Embodiment 48

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is SEQ ID NO:28.

Embodiment 49

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is SEQ ID NO:29.

Embodiment 50

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is SEQ ID NO:30.

Embodiment 51

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is SEQ ID NO:31.

Embodiment 52

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is SEQ ID NO:32.

Embodiment 53

The engineered polypeptide according to any one of Embodiments 1 to 20,wherein said HD1 is SEQ ID NO:33.

Embodiment 54

The engineered polypeptide according to any one of Embodiments 1 to 53,wherein said ABD comprises an albumin binding motif (ABM) that consistsof the amino acid sequence:

(SEQ ID NO: 34) GVSD X₅ YK X₈ X₉ I X₁₁ X₁₂ A X₁₄ TVEGV X₂₀ AL X₂₃X₂₄ X₂₅ Iwherein, independently of each other,

X₅ is selected from Y and F;

X₈ is selected from N, R and S;

X₉ is selected from V, I, L, M, F and Y;

X₁₁ is selected from N, S, E and D;

X₁₂ is selected from R, K and N;

X₁₄ is selected from K and R;

X₂₀ is selected from D, N, Q, E, H, S, R and K;

X₂₃ is selected from K, I and T;

X₂₄ is selected from A, S, T, G, H, L and D; and X₂₅ is selected from H,E and D.

Embodiment 55

The engineered polypeptide according to any one of Embodiments 1 to 54,wherein, independently of each other,

X₅ is Y;

X₈ is N;

X₂₃ is T or I;

X₂₄ is S or L; and

X₂₅ iS E or H.

Embodiment 56

The engineered polypeptide according to any one of Embodiments 1 to 55,wherein the albumin binding motif comprises an amino acid sequence thatis selected from the group consisting of: GVSDYYKNLINKAKTVEGVEALTLHI(SEQ ID NO:114) and GVSDYYKNLINKAKTVEGVEALISEI (SEQ ID NO:115).

Embodiment 57

The engineered polypeptide according to any one of Embodiments 1 to 56,wherein said ABD comprises an albumin binding motif (ABM) that is notGVSDYYKNLINNAKTVEGVKALIDEI (SEQ ID NO:35).

Embodiment 58

The engineered polypeptide according to any one of Embodiments 1 to 57,wherein said ABD comprises an ABM disclosed in Table 1.

Embodiment 59

The engineered polypeptide according to any one of Embodiments 1 to 58,wherein said ABD comprises the amino acid sequence:

(SEQ ID NO: 36) LAEAK X_(a) X_(b) A X_(c) X_(d) EL X_(e) KY-[ABM]-LAALPwherein

[ABM] is an albumin binding motif, and, independently of each other,X_(a) is selected from V and E;

X_(b) is selected from L, E and D;

X_(c) is selected from N, L and I;

X_(d) is selected from R and K;

X_(e) is selected from D and K;

the leucine at position 45 is present or absent; and the proline atposition 46 is present or absent.

Embodiment 60

The engineered polypeptide according to any one of Embodiments 1 to 59,wherein, independently of each other, X_(a) is E;

X_(b) is D;

X_(c) is I; and

X_(d) is K.

Embodiment 61

The engineered polypeptide according to any one of Embodiments 1 to 60,wherein the albumin binding domain polypeptide (ABD) comprises an aminoacid sequence that is selected from the group consisting of:

(SEQ ID NO: 50) LAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALTLHILAALP; and(SEQ ID NO: 51) LAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALISEILAALP.

Embodiment 62

The engineered polypeptide according to any one of Embodiments 1 to 61,wherein said ABD comprises the amino acid sequence:

(SEQ ID NO: 36) LAEAK X_(a) X_(b) A X_(c) X_(d) EL X_(e) KY-[ABM]-LAALPwherein

[ABM] is an albumin binding motif, and, independently of each other,X_(a) is selected from V and E;

X_(b) is selected from L, E and D;

X_(c) is selected from N, L and I;

X_(d) is selected from R and K;

X_(e) is selected from D and K; the leucine at position 45 is present orabsent;

the proline at position 46 is present or absent; and

wherein ABM consists of the amino acid sequence:

(SEQ ID NO: 34) GVSD X₅ YK X₈ X₉ I X₁₁ X₁₂ A X₁₄ TVEGV X₂₀ AL X₂₃X₂₄ X₂₅ Iwherein, independently of each other,

X₅ is selected from Y and F;

X₈ is selected from N, R and S;

X₉ is selected from V, I, L, M, F and Y;

X₁₁ is selected from N, S, E and D;

X₁₂ is selected from R, K and N;

X₁₄ is selected from K and R;

X₂₀ is selected from D, N, Q, E, H, S, R and K;

X₂₃ is selected from K, I and T;

X₂₄ is selected from A, S, T, G, H, L and D; and

X₂₅ is selected from H, E and D.

Embodiment 63

The engineered polypeptide according to any one of the Embodiments 1 to62, wherein said ABD comprises an amino acid sequence having at least85% identity with an amino acid sequence that is selected from the groupconsisting of SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40,SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45,SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50,SEQ ID NO:51, and SEQ ID NO:52.

Embodiment 64

The engineered polypeptide according to any one of Embodiments 1 to 63,wherein said ABD comprises any one of the peptides selected from thegroup consisting of:

(SEQ ID NO: 38) LAEAKVLANRELDKYGVSDFYKSYINRAKTVEGVHTLIGHILAALP,(SEQ ID NO: 39) LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVNALTHHILAALP,(SEQ ID NO: 40) LAEAKVLANRELDKYGVSDYYKNLINRARTVEGVHALIDHILAALP,(SEQ ID NO: 41) LAEAKVLANRELDKYGVSDYYKNIINRAKTVEGVRALKLHILAALP,(SEQ ID NO: 42) LAEAKVLANRELDKYGVSDFYKNLINRAKTVEGVSSLKGHILAALP,(SEQ ID NO: 43) LAEAKVLANRELDKYGVSDYYKNLINKAKTVEGVEALTLHILAALP,(SEQ ID NO: 44) LAEAKVLANRELDKYGVSDFYKNLINRAKTVEGVDALIAHILAALP,(SEQ ID NO: 45) LAEAKVLANRELDKYGVSDFYKSLINRAKTVEGVDALTSHILAALP,(SEQ ID NO: 46) LAEAKVLANRELDKYGVSDFYKNLINRAKTVEGVNSLTSHILAALP,(SEQ ID NO: 47) LAEAKVLANRELDKYGVSDFYKNVINKAKTVEGVEALIADILAALP,(SEQ ID NO: 48) LAEAKVLANRELDKYGVSDYYKNLINKAKTVEGVQALIAHILAALP,(SEQ ID NO: 49) LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP,(SEQ ID NO: 50) LAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALTLHILAALP,(SEQ ID NO:51) LAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALISEILAALP, and(SEQ ID NO: 52) LAEAKEDAIKELDKYGVSDYYKRLISKAKTVEGVKALISEILAALP.

Embodiment 65

The engineered polypeptide according to any one of Embodiments 1 to 64,wherein said linker L1 is a peptide of from 1 to 30 amino acids or lessthan 30 amino acids.

Embodiment 66

The engineered polypeptide according to any one of Embodiments 1 to 65,wherein said linker L1 is selected from the 20 naturally occurring aminoacids.

Embodiment 67

The engineered polypeptide according to any one of Embodiments 1 to 66,wherein said linker L1 is a non-natural amino acids incorporated bychemical synthesis, post-translational chemical modification or by invivo incorporation by recombinant expression in a host cell.

Embodiment 68

The engineered polypeptide according to any one of Embodiments 1 to 67,wherein said linker L1 amino acids are selected from serine, glycine,alanine, proline, asparagine, glutamine, glutamate, aspartate, andlysine.

Embodiment 69

The engineered polypeptide according to any one of Embodiments 1 to 68,wherein said linker L1 comprises a majority of amino acids that aresterically unhindered.

Embodiment 70

The engineered polypeptide according to any one of Embodiments 1 to 69,wherein said linker L1 comprises one or more of the following: an acidiclinker, a basic linker, and a structural motif Embodiment 71. Theengineered polypeptide according to any one of Embodiments 1 to 70,wherein said linker L1 comprises polyglycine, polyalanines,poly(Gly-Ala), or poly(Gly-Ser).

Embodiment 72

The engineered polypeptide according to any one of Embodiments 1 to 71,wherein said linker L1 comprises a polyglycine of (Gly)₃, (Gly)₄ (SEQ IDNO: 116), or (Gly)₅ (SEQ ID NO: 117).

Embodiment 73

The engineered polypeptide according to any one of Embodiments 1 to 72,wherein said linker L1 comprises (Gly)₃Lys(Gly)₄ (SEQ ID NO: 118);(Gly)₃AsnGlySer(Gly)₂ (SEQ ID NO: 119); (Gly)₃Cys(Gly)₄ (SEQ ID NO:120); and GlyProAsnGlyGly (SEQ ID NO: 121).

Embodiment 74

The engineered polypeptide according to any one of Embodiments 1 to 73,wherein said linker L1 comprises a combination of Gly and Ala.

Embodiment 75

The engineered polypeptide according to any one of Embodiments 1 to 74,wherein said linker L1 comprises a combination of Gly and Ser.

Embodiment 76

The engineered polypeptide according to any one of Embodiments 1 to 75,wherein said linker L1 comprises a combination of Gly and Glu.

Embodiment 77

The engineered polypeptide according to any one of Embodiments 1 to 76,wherein said linker L1 comprises a combination of Gly and Lys.

Embodiment 78

The engineered polypeptide according to any one of Embodiments 1 to 77,wherein said linker L1 comprises a sequence selected from groupconsisting of: [Gly-Ser]_(n) (SEQ ID NO: 122), [Gly-Gly-Ser]_(n) (SEQ IDNO: 123), [Gly-Gly-Gly-Ser]_(n) (SEQ ID NO: 124) and[Gly-Gly-Gly-Gly-Ser]_(n) (SEQ ID NO: 125); where n is 1, 2, 3, 4, 5, 6,7, 8, 9, or 10.

Embodiment 79

The engineered polypeptide according to any one of Embodiments 1 to 78,wherein said linker L1 comprises a sequence selected from the groupconsisting of: [Gly-Glu]_(n) (SEQ ID NO: 126); [Gly-Gly-Glu]_(n) (SEQ IDNO: 127); [Gly-Gly-Gly-Glu]_(n) (SEQ ID NO: 128);[Gly-Gly-Gly-Gly-Glu]_(n) (SEQ ID NO: 129), [Gly-Asp]_(n) (SEQ ID NO:130); [Gly-Gly-Asp]_(n) (SEQ ID NO: 131); [Gly-Gly-Gly-Asp]_(n) (SEQ IDNO: 132); [Gly-Gly-Gly-Gly-Asp]_(n) (SEQ ID NO: 133) where n is 1, 2, 3,4, 5, 6, 7, 8, 9, 10.

Embodiment 80

The engineered polypeptide according to any one of Embodiments 1 to 79,wherein said linker L1 comprises a sequence selected from the groupconsisting of:

(SEQ ID NO: 126) [Gly-Glu]_(n); (SEQ ID NO: 127) [Gly-Gly-Glu]_(n);(SEQ ID NO: 128) [Gly-Gly-Gly-Glu]_(n); (SEQ ID NO: 129)[Gly-Gly-Gly-Gly-Glu]_(n), (SEQ ID NO: 130) [Gly-Asp]_(n);(SEQ ID NO: 131) [Gly-Gly-Asp]_(n); (SEQ ID NO: 132)[Gly-Gly-Gly-Asp]_(n); (SEQ ID NO: 133) [Gly-Gly-Gly-Gly-Asp]_(n)where _(n) is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

Embodiment 81

The engineered polypeptide according to any one of Embodiments 1 to 80,wherein said linker L1 comprises a sequence selected from the groupconsisting of:

(SEQ ID NO: 134) [Gly-Lys]_(n); (SEQ ID NO: 135) [Gly-Gly-Lys]_(n);(SEQ ID NO: 136) [Gly-Gly-Gly-Lys]_(n); (SEQ ID NO: 137)[Gly-Gly-Gly-Gly-Lys]_(n), (SEQ ID NO: 138) [Gly-Arg]_(n);(SEQ ID NO: 139) [Gly-Gly-Arg]_(n); (SEQ ID NO: 140)[Gly-Gly-Gly-Arg]_(n); (SEQ ID NO: 141) [Gly-Gly-Gly-Gly-Arg]_(n)where _(n) is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

Embodiment 82

The engineered polypeptide according to any one of Embodiments 1 to 81,wherein said linker L1 comprises a sequence selected from the groupconsisting of: [Glu-Ala-Ala-Ala-Lys]_(n) (SEQ ID NO: 142), where n is 1,2, 3, 4, 5, 6, 7, 8, 9, 10.

Embodiment 83

The engineered polypeptide according to any one of Embodiments 1 to 81,wherein said linker L1 comprises a sequence selected from the groupconsisting of:

(SEQ ID NO: 153) [Gly-Gly-Glu]₆ (SEQ ID NO: 154) [Gly-Gly-Lys]₆.(SEQ ID NO: 155) [Glu-Ala-Ala-Ala-Lys]₃,, (SEQ ID NO: 156)[Glu-Ala-Ala-Ala-Lys]₄, or (SEQ ID NO: 157) [Glu-Ala-Ala-Ala-Lys]₅.

Embodiment 84

The engineered polypeptide according to any one of Embodiments 1 to 83,wherein said linker L1 comprises a N-terminal TG dipeptide.

Embodiment 85

The engineered polypeptide according to any one of Embodiments 1 to 84,wherein said linker L1 comprises a C-terminal AS dipeptide.

Embodiment 86

The engineered polypeptide according to any one of Embodiments 1 to 85,wherein said linker L1 comprises an N-terminal TG dipeptide and aC-terminal AS dipeptide.

Embodiment 87

The engineered polypeptide according to any one of Embodiments 1 to 86,wherein said linker L1 comprises an amino acids sequence that isselected from the group consisting of TG-(GGGS)₁ (SEQ ID NO: 215),TG-(GGGS)₂ (SEQ ID NO: 216), TG-(GGGS)₃ (SEQ ID NO: 217), TG-(GGGS)₄(SEQ ID NO: 218), TG-(GGGS)₅ (SEQ ID NO: 219), (GGGS)₁-AS (SEQ ID NO:220), (GGGS)₂-AS (SEQ ID NO: 221), (GGGS)₃-AS (SEQ ID NO: 222),(GGGS)₄-AS (SEQ ID NO: 223), (GGGS)₅-AS (SEQ ID NO: 224), TG-(GGGS)₁-AS(SEQ ID NO: 225), TG-(GGGS)₂-AS (SEQ ID NO: 226), TG-(GGGS)₃-AS (SEQ IDNO: 227), TG-(GGGS)₄-AS (SEQ ID NO: 228), and TG-(GGGS)₅-AS (SEQ ID NO:229).

Embodiment 88

The engineered polypeptide according to any one of Embodiments 1 to 87,wherein said TG dipeptide TG and/or said dipeptide AS are absent or arereplaced by a pair of amino acids selected from T, A, S, and G.

Embodiment 89

The engineered polypeptide according to any one of Embodiments 1 to 88,wherein said polypeptide further comprises one or more additionallinkers.

Embodiment 90

The engineered polypeptide according to any one of Embodiments 1 to 89,wherein said engineered polypeptide comprises an amino acid sequenceselected from the group consisting of: SEQ ID NO:53, SEQ ID NO:54, SEQID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ IDNO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ IDNO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ IDNO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ IDNO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ IDNO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ IDNO:85 SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ IDNO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ IDNO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ IDNO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQID NO:105, SEQ ID NO:106, and SEQ ID NO:107.

Embodiment 91

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:53.

Embodiment 92

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:54.

Embodiment 93

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:55.

Embodiment 94

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:56.

Embodiment 95

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:57.

Embodiment 96

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:58.

Embodiment 97

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:59.

Embodiment 98

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:60.

Embodiment 99

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:61.

Embodiment 100

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:62.

Embodiment 101

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:63.

Embodiment 102

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:64.

Embodiment 103

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:65.

Embodiment 104

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:66.

Embodiment 105

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:67.

Embodiment 106

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:68.

Embodiment 107

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:69.

Embodiment 108

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:70.

Embodiment 109

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:71.

Embodiment 110

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:72.

Embodiment 111

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:73.

Embodiment 112

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:74.

Embodiment 113

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:75.

Embodiment 114

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:76.

Embodiment 115

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:77.

Embodiment 116

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:78.

Embodiment 117

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:79.

Embodiment 118

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:80.

Embodiment 119

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:81.

Embodiment 120

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:82.

Embodiment 121

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:83.

Embodiment 122

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:84.

Embodiment 123

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:85.

Embodiment 124

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:86.

Embodiment 125

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:87.

Embodiment 126

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:88.

Embodiment 127

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:89.

Embodiment 128

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:90.

Embodiment 129

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:91.

Embodiment 130

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:92.

Embodiment 131

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:93.

Embodiment 132

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:94.

Embodiment 133

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:95.

Embodiment 134

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:96.

Embodiment 135

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:97.

Embodiment 136

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:98.

Embodiment 137

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:99.

Embodiment 138

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:100.

Embodiment 139

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:102.

Embodiment 140

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:102.

Embodiment 141

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:103.

Embodiment 142

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:104.

Embodiment 143

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:105.

Embodiment 144

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:106.

Embodiment 145

The engineered polypeptide according to any one of Embodiments 1 to 90,wherein said engineered polypeptide comprises the amino acid sequenceselected set out in SEQ ID NO:107.

Embodiment 146

The engineered polypeptide according to any one of Embodiments 1 to 145,having affinity for serum albumin with a dissociation constant less thanabout 10⁻⁶ mol/L.

Embodiment 147

The engineered polypeptide according to any one of Embodiments 1 to 146,having affinity for serum albumin with a dissociation constant less thanabout 10⁻⁹ mol/L.

Embodiment 148

The engineered polypeptide according to any one of Embodiments 1 to 147,having affinity for serum albumin with a dissociation constant less thanabout 10⁻¹² mol/L.

Embodiment 149

The engineered polypeptide according to any one of Embodiments 1 to 148,wherein the polypeptide has a duration of action of at least 1 day.

Embodiment 150

The engineered polypeptide according to any one of Embodiments 1 to 149,wherein the polypeptide has a duration of action of at least 3 days.

Embodiment 151

The engineered polypeptide according to any one of Embodiments 1 to 150,wherein the polypeptide has a duration of action of at least 5 days.

Embodiment 152

The engineered polypeptide according to any one of Embodiments 1 to 151,wherein the polypeptide has a duration of action of at least 5 days in ahuman subject.

Embodiment 153

A method for treating a disease or disorder in a subject, comprisingadministering a engineered polypeptide according to any one ofEmbodiments 1 to 152 and 170 to 192 to a subject in need thereof in anamount effective to treat said disease or disorder.

Embodiment 154

The method according to Embodiment 153, wherein said disease or disorderis disease or disorder can be lipodystrophy, dyslipidemia,hyperlipidemia, overweight, obesity, hypothalamic amenorrhea,Alzheimer's disease, leptin deficiency, fatty liver disease, diabetes(including type I and type II), nonalcoholic steatohepatitis (NASH),nonalcoholic fatty liver disease (NAFLD) and metabolic syndrome X.

Embodiment 155

The method according to Embodiment 153 or Embodiment 154, wherein saiddisease or disorder is lipodystrophy, dyslipidemia, hyperlipidemia,overweight, obesity, hypothalamic amenorrhea, Alzheimer's disease,leptin deficiency, fatty liver disease or diabetes.

Embodiment 156

The method according to any one of Embodiments 153 to 155, wherein saiddisease or disorder is type I diabetes or type II diabetes.

Embodiment 157

The method according to any one of Embodiments 153 to 155, wherein saiddisease or disorder is obesity.

Embodiment 158

The method according to any one of Embodiments 153 to 155, wherein saiddisease or disorder is lipodystrophy or leptin deficiency.

Embodiment 159

A pharmaceutical composition comprising an engineered polypeptideaccording to any one of Embodiments 1 to 152 and a pharmaceuticallyacceptable excipient.

Embodiment 160

The pharmaceutical composition according to Embodiment 159, wherein saidpharmaceutical composition is an injectable pharmaceutical composition.

Embodiment 161

The pharmaceutical composition according to any one of Embodiments 159to 160, wherein said pharmaceutical composition is a sustained releaseor long lasting pharmaceutical composition.

Embodiment 162

The pharmaceutical composition according to any one of Embodiments 159to 161, wherein said pharmaceutical composition is a once dailypharmaceutical composition.

Embodiment 163

The pharmaceutical composition according to any one of Embodiments 159to 161, wherein said pharmaceutical composition is a once weeklypharmaceutical composition.

Embodiment 164

A pharmaceutical composition of any one of Embodiments 159 to 163 fortreating a disease or disorder in a subject.

Embodiment 165

The pharmaceutical composition according to any one of Embodiments 159to 164 wherein the disease or disorder is lipodystrophy, dyslipidemia,hyperlipidemia, overweight, obesity, hypothalamic amenorrhea,Alzheimer's disease, leptin deficiency, fatty liver disease, diabetes(including type I and type II), nonalcoholic steatohepatitis (NASH),nonalcoholic fatty liver disease (NAFLD) and metabolic syndrome X.

Embodiment 166

The pharmaceutical composition of Embodiment 164 or Embodiment 165wherein said disease or disorder is lipodystrophy, dyslipidemia,hyperlipidemia, overweight, obesity, hypothalamic amenorrhea,Alzheimer's disease, leptin deficiency, fatty liver disease or diabetes.

Embodiment 167

The method according to any one of Embodiments 164 to 166, wherein saiddisease or disorder is type I diabetes or type II diabetes.

Embodiment 168

The method according to any one of Embodiments 164 to 166, wherein saiddisease or disorder is obesity.

Embodiment 169

The method according to any one of Embodiments 164 to 166, wherein saiddisease or disorder is lipodystrophy or leptin deficiency.

Embodiment 170

The engineered polypeptide according to any one of embodiments 1 to 18,wherein said HD1 is selected from the group consisting of:

-   -   (a) the amino acid sequence 1-146 of a leptin selected from the        group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ        ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ        ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13,        SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID        NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22,        SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID        NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31,        SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143, SEQ ID NO:144, SEQ ID        NO:145, and SEQ ID NO:146; in which a different amino acid is        substituted in one or more of the following positions and        retaining the same numbering (even in the absence of a        glutaminyl residue at position 28): 4, 32, 33, 35, 50, 64, 68,        71, 74, 77, 78, 89, 97, 100, 102, 105, 106, 107, 108, 111, 118,        136, 138, 142, and 145;    -   (b) the amino acid sequence of subpart (a) in which the        glutaminyl residue at position 28 is absent;    -   (c) the amino acid sequence of subparts (a) or (b) in which a        methionyl residue is added at the N-terminus;    -   (d) a leptin consisting of a fragment of the amino acid sequence        of (a), (b), or (c) selected from the group consisting of:        -   (i) amino acids 98-146;        -   (ii) amino acids 1-32;        -   (iii) amino acids 40-116;        -   (iv) amino acids 1-99 and 112-146;        -   (v) amino acids 1-99 and 112-146 in which one or more of            amino acids 100-111 is placed between amino acids 99 and            112;        -   (vi) the amino acid sequence of subpart (i) wherein one or            more of amino acids 100, 102, 105, 106, 107, 108, 111, 118,            136, 138, 142, and 145 is substituted with another amino            acid;        -   (vii) the amino acid sequence of subpart (ii) wherein one or            more of amino acids 4, 8 and 32 is substituted with another            amino acid;        -   (viii) the amino acid sequence of subpart (iii) wherein one            or more of amino acids 50, 53, 60, 64, 66, 67, 68, 71, 74,            77, 78, 89, 97, 100, 102, 105, 106, 107, 108, 111 and 112 is            replaced with another amino acid;        -   (ix) the amino acid sequence of subpart (iv) wherein one or            more of amino acids 4, 8, 32, 33, 35, 48, 50, 53, 60, 64,            66, 67, 68, 71, 74, 77, 78, 89, 97, 112, 118, 136, 138, 142,            and 145 is replaced with another amino acid; and        -   (x) the amino acid sequence of subpart (v) wherein one or            more of amino acids 4, 32, 33, 35, 50, 64, 68, 71, 74, 77,            78, 89, 97, 100, 102, 105, 106, 107, 108, 111, 118, 136,            138, 142, and 145 is replaced with another amino acid;    -   (xi) the amino acid sequence of any of subparts (i)-(x) wherein        a methionine has been added at the N-terminus;    -   (e) the amino acid sequence of any of subparts (a) through (d)        wherein said amino acid sequence is attached to a chemical        moiety;    -   (f) the amino acid sequence of subpart (e) wherein said chemical        moiety is a water soluble polymer moiety;    -   (g) the amino acid sequence of subpart (f) wherein said water        soluble polymer moiety is selected from the group consisting of:        polyethylene glycol, an ethylene glycol/propylene glycol        copolymer, a carboxymethylcellulose, a dextran, a polyvinyl        alcohol, a polyvinyl pyrolidone, a poly-1,3-dioxolane, a        poly-1,3,6-trioxane, an ethylene/maleic anhydride copolymer, a        polyaminoacid homopolymer, a polyaminoacid random copolymer, an        albumin, an Fc protein, a poly(n-vinyl pyrolidone)polyethylene        glycol, a propylene glycol homopolymer, a polypropylene        oxide/ethylene oxide copolymer, a polyoxyethylated polyol, a        polyvinyl alcohol, a polyethylene glycol propionadehyde, a        succinate, and a styrene;    -   (h) the amino acid sequence of subpart (g) wherein said water        soluble polymer moiety is a polyethylene glycol; and    -   (i) the amino acid sequence of subpart (g) wherein said water        soluble polymer is a polyamino acid selected from the group        consisting of: an albumin, an antibody, an Fc protein, and a        polylysine moiety.

Embodiment 171

The engineered polypeptide according to any one of embodiments 1 to 18and 170, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146; wherein one or moreamino acid substitutions have been made.

Embodiment 172

The engineered polypeptide according to any one of embodiments 1 to 18and 171, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146; wherein one amino acidsubstitution has been made.

Embodiment 173

The engineered polypeptide according to any one of embodiments 1 to 18and 171, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146; wherein two amino acidsubstitutions have been made.

Embodiment 174

The engineered polypeptide according to any one of embodiments 1 to 18and 171, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146; wherein three aminoacid substitutions have been made.

Embodiment 175

The engineered polypeptide according to any one of embodiments 1 to 18and 171, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146; wherein four amino acidsubstitutions have been made.

Embodiment 176

The engineered polypeptide according to any one of embodiments 1 to 18and 171, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146; wherein five amino acidsubstitutions have been made.

Embodiment 177

The engineered polypeptide according to any one of embodiments 1 to 18and 171, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146; wherein six amino acidsubstitutions have been made.

Embodiment 178

The engineered polypeptide according to any one of embodiments 1 to 18and 171, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146; wherein seven aminoacid substitutions have been made.

Embodiment 179

The engineered polypeptide according to any one of embodiments 1 to 18and 171, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146; wherein eight aminoacid substitutions have been made.

Embodiment 180

The engineered polypeptide according to any one of embodiments 1 to 18and 171, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146; wherein nine amino acidsubstitutions have been made.

Embodiment 181

The engineered polypeptide according to any one of embodiments 1 to 18and 171, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146; wherein ten amino acidsubstitutions have been made.

Embodiment 182

The engineered polypeptide according to any one of embodiments 1 to 18and 171, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146; wherein 11 amino acidsubstitutions have been made.

Embodiment 183

The engineered polypeptide according to any one of embodiments 1 to 18and 171, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146; wherein 12 amino acidsubstitutions have been made.

Embodiment 184

The engineered polypeptide according to any one of embodiments 1 to 18and 171, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146; wherein 13 amino acidsubstitutions have been made.

Embodiment 185

The engineered polypeptide according to any one of embodiments 1 to 18and 171, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146; wherein 14 amino acidsubstitutions have been made.

Embodiment 186

The engineered polypeptide according to any one of embodiments 1 to 18and 171, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146; wherein 15 amino acidsubstitutions have been made.

Embodiment 187

The engineered polypeptide according to any one of embodiments 1 to 18and 171, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146; wherein 16 amino acidsubstitutions have been made.

Embodiment 188

The engineered polypeptide according to any one of embodiments 1 to 18and 171, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146; wherein 17 amino acidsubstitutions have been made.

Embodiment 189

The engineered polypeptide according to any one of embodiments 1 to 18and 171, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146; wherein 18 amino acidsubstitutions have been made.

Embodiment 190

The engineered polypeptide according to any one of embodiments 1 to 18and 171, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146; wherein 19 amino acidsubstitutions have been made.

Embodiment 191

The engineered polypeptide according to any one of embodiments 1 to 18and 171, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146; wherein 20 amino acidsubstitutions have been made.

Embodiment 192

The engineered polypeptide according to any one of embodiments 1 to 18and 171, wherein said HD1 comprises an amino acid sequence selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143,SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146; wherein 21 amino acidsubstitutions have been made.

Embodiment 193

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:143.

Embodiment 194

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:144.

Embodiment 195

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:145.

Embodiment 196

The engineered polypeptide according to any one of embodiments 1 to 20,wherein said HD1 is SEQ ID NO:146.

While the foregoing description discloses the present invention, withexamples provided for the purpose of illustration, it will be understoodthat the practice of the present invention encompasses all of the usualvariations, adaptations, or modifications as being within the scope ofthe claimed invention. Therefore, descriptions and examples should notbe construed as limiting the scope of the invention, which is delineatedby the appended claims.

What is claimed is:
 1. An engineered polypeptide comprising: an albuminbinding domain polypeptide (ABD) and a first peptide hormone domain(HD1) selected from a leptin, a leptin analog or an active fragmentthereof.
 2. The engineered polypeptide according to claim 1, furthercomprising a first linker (L1) covalently linked to said HD1.
 3. Theengineered polypeptide according to claim 1, wherein said engineeredpolypeptide comprises said ABD as an N-terminal moiety and said HD1 as aC-terminal moiety.
 4. The engineered polypeptide according to claim 1,wherein said engineered polypeptide comprises said ABD as a C-terminalmoiety and said HD1 as an N-terminal moiety.
 5. The engineeredpolypeptide according to claim 1, wherein said HD1 has at least 50%identity with an amino acid sequence selected from the group consistingof: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, and SEQ ID NO:12. SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ IDNO:31, SEQ ID NO:32, SEQ ID NO:33.
 6. The engineered polypeptideaccording to claim 1, wherein said ABD comprises an albumin bindingmotif (ABM) that consists of the amino acid sequence: (SEQ ID NO: 34)GVSD X₅ YK X₈ X₉ I X₁₁ X₁₂ A X₁₄ TVEGV X₂₀ AL X₂₃ X₂₄ X₂₅ I

wherein, independently of each other, X₅ is selected from Y and F; X₈ isselected from N, R and S; X₉ is selected from V, I, L, M, F and Y;X_(1i) is selected from N, S, E and D; X₁₂ is selected from R, K and N;X₁₄ is selected from K and R; X₂₀ is selected from D, N, Q, E, H, S, Rand K; X₂₃ is selected from K, I and T; X₂₄ is selected from A, S, T, G,H, L and D; and X₂₅ is selected from H, E and D.
 7. The engineeredpolypeptide according to claim 6, wherein, independently of each other,X₅ is Y; X₈ is N; X₂₃ is T or I; X₂₄ is S or L; and X₂₅ is E or H. 8.The engineered polypeptide according to claim 7, wherein the albuminbinding motif comprises an amino acid sequence that is selected from thegroup consisting of: (SEQ ID NO: 114) GVSDYYKNLINKAKTVEGVEALTLHI and(SEQ ID NO: 115) GVSDYYKNLINKAKTVEGVEALISEI.


9. The engineered polypeptide according to claim 1, wherein said ABDcomprises an albumin binding motif (ABM) that is notGVSDYYKNLINNAKTVEGVKALIDEI (SEQ ID NO:35).
 10. The engineeredpolypeptide according to claim 2, wherein the albumin binding domainpolypeptide (ABD) comprises an amino acid sequence that is selected fromthe group consisting of: (SEQ ID NO: 50)LAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALTLHILAALP; and (SEQ ID NO: 51)LAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALISEILAALP.


11. The engineered polypeptide according to claim 1, wherein said ABDcomprises the amino acid sequence: (SEQ ID NO: 36)LAEAK X_(a) X_(b) A X_(c) X_(d) EL X_(e) KY-[ABM]-LAALP

wherein [ABM] is an albumin binding motif, and, independently of eachother, X_(a) is selected from V and E; X_(b) is selected from L, E andD; X_(e) is selected from N, L and I; X_(d) is selected from R and K;X_(e) is selected from D and K; the leucine at position 45 is present orabsent; the proline at position 46 is present or absent; and wherein ABMconsists of the amino acid sequence: (SEQ ID NO: 34)GVSD X₅ YK X₈ X₉ I X₁₁ X₁₂ A X₁₄ TVEGV X₂₀ AL X₂₃ X₂₄ X₂₅ I

wherein, independently of each other, X₅ is selected from Y and F; X₈ isselected from N, R and S; X₉ is selected from V, I, L, M, F and Y; X₁₁is selected from N, S, E and D; X₁₂ is selected from R, K and N; X₁₄ isselected from K and R; X₂₀ is selected from D, N, Q, E, H, S, R and K;X₂₃ is selected from K, I and T; X₂₄ is selected from A, S, T, G, H, Land D; and X₂₅ is selected from H, E and D.
 12. The engineeredpolypeptide according to claim 11, wherein said ABD comprises an aminoacid sequence having at least 85% identity with an amino acid sequencethat is selected from the group consisting of SEQ ID NO: 37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ IDNO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ IDNO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, and SEQ ID NO:52. 13.The engineered polypeptide according to claim 12, wherein said ABDcomprises any one of the peptides selected from the group consisting of:(SEQ ID NO: 38) LAEAKVLANRELDKYGVSDFYKSYINRAKTVEGVHTLIGHILAALP,(SEQ ID NO: 39) LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVNALTHHILAALP,(SEQ ID NO: 40) LAEAKVLANRELDKYGVSDYYKNLINRARTVEGVHALIDHILAALP,(SEQ ID NO: 41) LAEAKVLANRELDKYGVSDYYKNIINRAKTVEGVRALKLHILAALP,(SEQ ID NO: 42) LAEAKVLANRELDKYGVSDFYKNLINRAKTVEGVSSLKGHILAALP,(SEQ ID NO: 43) LAEAKVLANRELDKYGVSDYYKNLINKAKTVEGVEALTLHILAALP,(SEQ ID NO: 44) LAEAKVLANRELDKYGVSDFYKNLINRAKTVEGVDALIAHILAALP,(SEQ ID NO: 45) LAEAKVLANRELDKYGVSDFYKSLINRAKTVEGVDALTSHILAALP,(SEQ ID NO: 46) LAEAKVLANRELDKYGVSDFYKNLINRAKTVEGVNSLTSHILAALP,(SEQ ID NO: 47) LAEAKVLANRELDKYGVSDFYKNVINKAKTVEGVEALIADILAALP,(SEQ ID NO: 48) LAEAKVLANRELDKYGVSDYYKNLINKAKTVEGVQALIAHILAALP,(SEQ ID NO: 49) LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP,(SEQ ID NO: 50) LAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALTLHILAALP,(SEQ ID NO: 51) LAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALiseILAALP, and(SEQ ID NO: 52) LAEAKEDAIKELDKYGVSDYYKRLISKAKTVEGVKALISEILAALP.


14. The engineered polypeptide according to claim 2, wherein said linkerL1 is a peptide of from 1 to 30 amino acids.
 15. The engineeredpolypeptide according to claim 14, wherein said linker L1 comprises aN-terminal TG dipeptide.
 16. The engineered polypeptide according toclaim 14, wherein said linker L1 comprises a C-terminal AS dipeptide.17. The engineered polypeptide according to claim 14, wherein saidlinker L1 comprises an N-terminal TG dipeptide and a C-terminal ASdipeptide.
 18. The engineered polypeptide according to claim 14, whereinsaid linker L1 comprises an amino acids sequence that is selected fromthe group consisting of TG-(GGGS)₁ (SEQ ID NO: 215), TG-(GGGS)₂ (SEQ IDNO: 216), TG-(GGGS)₃ (SEQ ID NO: 217), TG-(GGGS)₄ (SEQ ID NO: 218),TG-(GGGS)₅ (SEQ ID NO: 219), (GGGS)₁-AS (SEQ ID NO: 220), (GGGS)₂-AS(SEQ ID NO: 221), (GGGS)₃-AS (SEQ ID NO: 222), (GGGS)₄-AS (SEQ ID NO:223), (GGGS)₅-AS (SEQ ID NO: 224), TG-(GGGS)₁-AS (SEQ ID NO: 225),TG-(GGGS)₂-AS (SEQ ID NO: 226), TG-(GGGS)₃-AS (SEQ ID NO: 227),TG-(GGGS)₄-AS (SEQ ID NO: 228), and TG-(GGGS)₅-AS (SEQ ID NO: 229). 19.The engineered polypeptide according to claim 1, wherein saidpolypeptide further comprises one or more additional linkers.
 20. Theengineered polypeptide according to claim 1, wherein said engineeredpolypeptide comprises an amino acid sequence selected from the groupconsisting of: SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56,SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61,SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66,SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71,SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76,SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81,SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85 SEQ ID NO:86, SEQID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ IDNO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ IDNO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ IDNO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, andSEQ ID NO:107.
 21. A method for treating a disease or disorder in asubject, comprising administering a engineered polypeptide according toclaim 1 to a subject in need thereof in an amount effective to treatsaid disease or disorder.
 22. The method according to claim 21, whereinsaid disease or disorder is disease or disorder can be lipodystrophy,dyslipidemia, hyperlipidemia, overweight, obesity, hypothalamicamenorrhea, Alzheimer's disease, leptin deficiency, fatty liver disease,diabetes, type I diabetes, type II diabetes, nonalcoholicsteatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD),metabolic syndrome X and Huntington's Disease.
 23. The method accordingto claim 22, wherein said disease or disorder is lipodystrophy,dyslipidemia, hyperlipidemia, overweight, obesity, hypothalamicamenorrhea, Alzheimer's disease, leptin deficiency, fatty liver diseaseor diabetes.
 24. A pharmaceutical composition comprising an engineeredpolypeptide according to claim 1 and a pharmaceutically acceptableexcipient.
 25. The pharmaceutical composition according to claim 24,wherein said pharmaceutical composition is formulated as an injectablepharmaceutical composition.
 26. The pharmaceutical composition accordingto claim 24, wherein said pharmaceutical composition is formulated as asustained release or long lasting pharmaceutical composition.
 27. Thepharmaceutical composition according to claim 24 formulated for use inthe treatment of lipodystrophy, dyslipidemia, hyperlipidemia,overweight, obesity, hypothalamic amenorrhea, Alzheimer's disease,leptin deficiency, fatty liver disease, diabetes (including type I andtype II), nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liverdisease (NAFLD) and metabolic syndrome X.
 28. The engineered polypeptideaccording to claim 1, wherein said HD1 is selected from the groupconsisting of: (a) the amino acid sequence 1-146 of a leptin selectedfrom the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:143, SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146; in which adifferent amino acid is substituted in one or more of the followingpositions and retaining the same numbering (even in the absence of aglutaminyl residue at position 28): 4, 32, 33, 35, 50, 64, 68, 71, 74,77, 78, 89, 97, 100, 102, 105, 106, 107, 108, 111, 118, 136, 138, 142,and 145; (b) the amino acid sequence of subpart (a) in which theglutaminyl residue at position 28 is absent; (c) the amino acid sequenceof subparts (a) or (b) in which a methionyl residue is added at theN-terminus; (d) a leptin consisting of a fragment of the amino acidsequence of (a), (b), or (c) selected from the group consisting of: (i)amino acids 98-146; (ii) amino acids 1-32; (iii) amino acids 40-116;(iv) amino acids 1-99 and 112-146; (v) amino acids 1-99 and 112-146 inwhich one or more of amino acids 100-111 is placed between amino acids99 and 112; (vi) the amino acid sequence of subpart (i) wherein one ormore of amino acids 100, 102, 105, 106, 107, 108, 111, 118, 136, 138,142, and 145 is substituted with another amino acid; (vii) the aminoacid sequence of subpart (ii) wherein one or more of amino acids 4, 8and 32 is substituted with another amino acid; (viii) the amino acidsequence of subpart (iii) wherein one or more of amino acids 50, 53, 60,64, 66, 67, 68, 71, 74, 77, 78, 89, 97, 100, 102, 105, 106, 107, 108,111 and 112 is replaced with another amino acid; (ix) the amino acidsequence of subpart (iv) wherein one or more of amino acids 4, 8, 32,33, 35, 48, 50, 53, 60, 64, 66, 67, 68, 71, 74, 77, 78, 89, 97, 112,118, 136, 138, 142, and 145 is replaced with another amino acid; and (x)the amino acid sequence of subpart (v) wherein one or more of aminoacids 4, 32, 33, 35, 50, 64, 68, 71, 74, 77, 78, 89, 97, 100, 102, 105,106, 107, 108, 111, 118, 136, 138, 142, and 145 is replaced with anotheramino acid; (xi) the amino acid sequence of any of subparts (i)-(x)wherein a methionine has been added at the N-terminus; (e) the aminoacid sequence of any of subparts (a) through (d) wherein said amino acidsequence is attached to a chemical moiety; (f) the amino acid sequenceof subpart (e) wherein said chemical moiety is a water soluble polymermoiety; (g) the amino acid sequence of subpart (f) wherein said watersoluble polymer moiety is selected from the group consisting of:polyethylene glycol, an ethylene glycol/propylene glycol copolymer, acarboxymethylcellulose, a dextran, a polyvinyl alcohol, a polyvinylpyrolidone, a poly-1,3-dioxolane, a poly-1,3,6-trioxane, anethylene/maleic anhydride copolymer, a polyaminoacid homopolymer, apolyaminoacid random copolymer, an albumin, an Fc protein, apoly(n-vinyl pyrolidone)polyethylene glycol, a propylene glycolhomopolymer, a polypropylene oxide/ethylene oxide copolymer, apolyoxyethylated polyol, a polyvinyl alcohol, a polyethylene glycolpropionadehyde, a succinate, and a styrene; (h) the amino acid sequenceof subpart (g) wherein said water soluble polymer moiety is apolyethylene glycol; and (i) the amino acid sequence of subpart (g)wherein said water soluble polymer is a polyamino acid-selected from thegroup consisting of: an albumin, an antibody, an Fc protein, and apolylysine moiety.
 29. The engineered polypeptide according to claim 28,wherein said HD1 comprises an amino acid sequence selected from thegroup consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143, SEQ IDNO:144, SEQ ID NO:145, and SEQ ID NO:146; wherein one or more amino acidsubstitutions have been made.
 30. The engineered polypeptide accordingto claim 1, wherein said HD1 is SEQ ID NO:143, SEQ ID NO:144, SEQ IDNO:145 or SEQ ID NO:146.
 31. A method of treating obesity in a subjectcomprising peripherally administering therapeutically effective amountsof at least two different anti-obesity agents, wherein at least oneanti-obesity agent is an amylin, an amylin analog, an amylin agonist, oran amylin derivative (i.e. an amylin agent) and at least oneanti-obesity agent is an engineered polypeptide comprising: an albuminbinding domain polypeptide (ABD); and a first peptide hormone domain(HD1) selected from a leptin, a leptin analog or an active fragmentthereof.
 32. A method of reducing body weight in a subject comprisingperipherally administering therapeutically effective amounts of at leasttwo different anti-obesity agents, wherein at least one anti-obesityagent is an amylin, an amylin analog, an amylin agonist, or an amylinderivative (i.e. an amylin agent) and at least one anti-obesity agent isan engineered polypeptide comprising: an albumin binding domainpolypeptide (ABD); and a first peptide hormone domain (HD1) selectedfrom a leptin, a leptin analog or an active fragment thereof.